MXPA97004765A - Improvements in or that are related to the endometr function - Google Patents

Abstract

The present invention relates to the use of a nucleic acid in the preparation of a medicament for transfecting at least some of the cells of the reproductive tract of a human female mammal by introducing into the cells a nucleic acid.

Description

IMPROVEMENTS IN OR THAT ARE RELATED TO THE ENDOMETRIAL FUNCTION Field of the Invention This invention relates, inter alia, to a method for altering one or more characteristics of endometrial tissue of mammals.

BACKGROUND OF THE INVENTION Endometrial physiology Two main cases are required for the embryo to be fixed in the uterus of the mammal; First, the preparation of the endometrium in such a way that it is receptive to the presence of a blastocyst that can then be implanted and acquire a nutritive support through the formation of the placenta and, secondly, the modification of the ac Myometrium must become quiescent, and remain so that the blastocyst becomes a resident within the uterine cavity, without danger of being expelled. Both cases are controlled by the action of the hormones of em barazo, whose estrogens and progesterone are particularly important. These steroid hormones act on the trio endome and the myometrium through their receptors, which are located in the nucleus of the target cells. Once activated, the steroid-nuclear receptor complex interacts with specific regions within the DNA to stimulate, repress or depress the genes encoding proteins and polypeptides such as enzymes or growth factors. The initiation of implantation is produced by a cascade of biochemical or biophysical changes. Adhesion molecules (eg CAM 105) have been implicated in the early stages of blastocyte fixation to the wall of the uterus. Later, the blastocyst and the endometrium take several stratagems to improve intimacy between the fetal and maternal tissues. In the ungulate trophoblast cells that form the outermost layer of the blastocyst, they migrate to the epithelium with which they subsequently fuse. The migration of the cells is produced by an additional stage in the woman, because not only the types of isolated or specific cell migrate, but large areas of trophoblase that insinuate themselves between the uterine epithelial cells. In order to do this, some of the trophoblast cells are fused together to form a syncytium. The procedure is very fast and the embryo becomes fixed in the uterine tissues without much apparent degeneration in the uterine epithelium. In some specimens, the implantation process is delayed either to wait for indications of the environment that assure birth at favorable moments of time, or -by means of physiological factors such as lactation, -so that the mother has finished weaning at delivery - before the next pregnancy is fully established. The preparation of the uterus for implantation is regulated by the secretion of ovarian hormones. The transport of the fertilized ovum through the oviduct must be precisely timed so that it reaches the uterus at the correct time of development and when it is in an adequate condition to receive it. Under most conditions, the uterus is hostile to the embryo, in fact, more hostile than some other parts of the body. The uterine lining of the uterus, under most conditions, is resistant to union and invasion by the trophoblast, and only under very precise hormonal conditions is this resistance relaxed. In unpaired mice and rats, animals do not have a complete cycle of brama because they do not form a normal secretory corpus luteum that produces increased amounts of progesterone. If mating takes place in the brama, a moment when high levels of estrogen are secreted by the ovarian follicle, from which the ovum is detached, a corpus luteum will form in the place of the broken follicle, producing concentrations of progesterone. they are then secreted, the implantation takes place and the pregnancy progresses (duration 21 days). Yes: there is an infertile copulation, similar cases take place except that the corpus luteum lasts only about 11 days and the pseudopregnancy is cut off. The cellular and biochemical changes that take place in the endometrium, for the most part, have been conscientiously studied in the mouse and in the rat, even though the information regarding these aspects in women has increased substantially in recent years. The endometrium in all species is made up of three main tissues - lu minal epithelium, glandular epithelium and stroma. The proliferation of cells takes place at different times in the three tissues. Luteinal cells proliferate immediately before estrus (proestrus) under the influence of the rising levels of estrogen produced by the follicles in the ovary. On day 1 of pregnancy (day of copulation in rodents) they have given up the division, but then experience a second, although smaller, activity impulse on day 3. The glandular cells show the highest activity on day 4 and then declines Stromal cells do not proliferate until day 4 but then, under the influence of progesterone, they reach high levels of proliferation on day 5. In women, fewer of these changes are known that foreshadow the implantation process, but this seems to be the maximum proliferation in the epithelial cells during the follicular phase of the cycle and the etromal cells during the luteal or secretory phase, as in the mouse and in the rat. The purpose of the endometriotic cell proliferation is not completely resolved. It is believed that to prepare the endometrium for implantation, increasing the number of cells that will serve a nutritive and secretory function (glandular epithelium) and that precipitate in the very early stages of the placentation (decidualization). As a prerequisite for successful implantation, the mitosis of the cell can progress towards cell differentiation and, therefore, play a crucial role in the initial events of the establishment of pregnancy. Evidence in support of this role is the endometrial production of growth factors (mitogens), cytokines and nuclear oncogenes. Some of these compounds are produced in increased concentrations in response to ovarian hormones that act through their receptors. Among the factors of growth, much attention is paid, usually to the epidermal growth factor (EGF), to the epidemiological growth factor that binds heparin (HBEGF), growth factors that agglutinate amfiregulin and insolin ( IGF-I 'and IGF-II). Evidence for the importance of the local action (paracrine) of at least one of these growth factors, the amfiregulin, has been provided by recent experiments in mice. The inhibition of the specific gene of implantation and regulated by progesterone for amhregulin was achieved by means of anti-progestin, RU486, and this resulted in the prevention of implantation (Das et al., Molecular Endo crimonology 0, 691-705 , nineteen ninety five). Among the cytokines, leukemia inhibitory factor (LIF) and colony stimulating factor (DSF), which are also produced by the mouse uterus at the time of implantation, have been found by studies, which -poses outside of combat the gene as indispensable, demonstrating that its removal is incompatible with implantation and normal pentation (Stewart et al., Nature 359, 76-79, 1992; Pollard et al., Developmental Biology 148, 273-283, 1991). Among the nuclear oncogenes levels of c-jun and c-fos (which are initially indicators of gene transcription) increase in the uterus after the administration of estrogen, and are inhibited by progesterone. There are important differences among the various species in the degree of invasion of the trophoblast at the time of implantation. In women, the initial trophoblast is highly invasive, whereas in pigs, which have a non-invasive form of implantation, the endometral epithelium is never branched through the three-month management period. The failure of the implantation in these species is high, reaching approximately 60% and 30%, respectively. The reasons for this high failure regime are complex and incompletely understood. In women, approximately half of the loss is attributable to genetic abnormalities but in pigs, as in other ungulates, where the loss is too high, genetic defects only account for a small percentage of the total. After failure of implantation in women, a drop in progesterone secretion causes bleeding, as in the end of the normal menstrual cycle; This does not have - most other animals. Disorders of menstruation, as well as implantation, are common. Furthermore, menstrual bleeding, either as a consequence of sequential hormone therapy, or together with continuous combined hormone replacement therapy or progestin-only late-acting contraceptives, is a significant cause of poor health in the woman. The priority reason - for this bleeding, or the reasons, are the focus of many current studies in biochemistry (For example prostaglandins, enzymes, polypeptides and proteins, vasoactive compounds such as platelet activating factor PAF, and vascular endothelial growth factor VEGF) and cellular mechanisms for example migrating cells that return to the uterus that produce the immunosuppressive compounds). A normal understanding of the reproductive processes focuses, largely, on the control of the reproduction of the steroidal hormone and the actions of these hormones on their target tissues. However, paracrine and autocrine factors are increasingly observed, which are key e-diators of reproductive function, although they interact with steroids (Benton, 1991 Current Opinion in Cell Biology 3_, 171-175; Rozengurt, 1993 Current Opinion in Cell Biology, 161-165; Tartakovsky et al., 1991 Developmental Biology 146, 345-352; Robertson et al., 1993 Current Opinion in Immunology 4, 585-590; Smith, 1994 Human Reproduction 9_, 936-946; and Tabibzadeh , 1994 Human Reproduction 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 the anti-TGFalpha antibody (TGF is "transforming growth factor") suggesting that the mitogenic effects of estrogen in this tissue are mediated by TGFalfa (Nelson et al., 1992 Endocrinology 151, 1657- 1664). Consequently, medical intervention in Gynecology is largely based on the steroidal / antero-uterine regulation of the uterus (Yen &Jaffe, 1991 in "Reproductive Endocrinology", Editors Ye, Jaffe &Benton, Pub. B Saunders, Philadelphia; Baird , 1993 British Medical Bulletin 49, 73-87). Despite the undoubted good result of this approach, conceptual advances in contraceptive technology have not been achieved for 20 years, no means to improve implantation, no progress made in providing placental growth and development, and no new contributions found. to treat gynecological diseases be nignas (menstrual dysfunction and fibroids). A number of publications have been made regarding 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 how to try "gene therapy" of humans by introducing nucleic acid sequences into the container, with the aim of overcoming a genetic deficiency in the recipient by expressing the polypeptides coded by the introduced nucleic acid sequences. -you give. Gene therapy tests have been conducted, for example, in which DNA sequences (incorporated within viral vectors) were introduced into the respiratory tracts of patients with cystic fibrosis, to alter -the phenotype of at least some of them. of the epithelial cells that cover the respiratory tract of patients. So far, attempts to introduce DNA into the endometrium of mammals have not been published, despite the availability of suitable techniques for this.

THE INVENTION In one aspect, the invention provides a method for altering one or more characteristics of at least some of the cells of the reproductive tract of an individual mammal by introducing a nucleic acid into the cells. In a second aspect, the invention provides a composition comprising nucleic acid for use in the alteration of one or more characteristics of at least some of the cells of the reproductive tract of a mammal. In a third aspect, the invention provides the use of a composition comprising nucleic acid to alter one or more characteristics of at least some of the cells of the reproductive tract of a mammal. In a fourth aspect, the invention provides the use of a composition comprising nucleic acid in the preparation of a substance to alter one or more characteristics of at least some of the cells of the reproductive tract of a mammal. In a fifth aspect , the invention provides a method for making a composition for use in the alteration of one or more characteristics of at least some of the cells in the reproductive tract of a mammal, comprising mixing a nucleic acid with a physiologically acceptable carrier.

The present invention can in no way be considered as an obvious extension of the germ therapy techniques already known as at least partially successful when applied to the lungs of patients with cystic fibrosis. The inherited genetic disorders are not believed to be responsible for any of the known diseases of the endometrium, so it may not have been the intention of those skilled in the art to apply the techniques of gene therapy to the endometrium. In addition, the epithelium of the endometrium is of a different type (cuboidal, derived from the colémic epithelium) compared to the epithelium of the lung (which is stratified) and, therefore, could not have been predicted to behave analagaly. Furthermore, at least in primates, there is detachment of the endometrial epithelium which could tend to cause the loss of any transferred cells. Finally, the inventors have found that there was no transfer of the DNA introduced into the organs of the mother, nor into the placenta of the embryo, any of which may have taken place and may have caused practical difficulties. Typically, the nucleic acid is introduced into a female mammal (preferably a female and, in particular, into its endometrial cells) Conveniently, the nucleic acid is introduced into the endometrial genital epithelium The nucleic acid can encode a polypeptide that It is already synthesized naturally by the cells - within which the nucleic acid is introduced, in such a way that the level of expression of that polypeptide is increased by a dosage effect of the gene. The method can be used to introduce the cells to express a polypeptide not previously synthesized in these cells, For example, the polypeptide can be an "artificial" polypeptide-dorecombinant that does not exist in nature, such as a chimeric polypeptide comprising, total or in part, functional domains of two or more different proteins.Nucleic acid is, preferably, DNA, but one can t to try to introduce RNA (either meaningful or nonsense fibers). An antisense molecule can be used to inhibit or otherwise interfere with the expression of a polypeptide in the cells within which the nucleic acid is introduced. The introducted nucleic acid sequence can be antisense RNA, or it can be a DNA sequence that directs the synthesis, intracellularly, of anti-sense RNA. Another way to achieve this inhibition is to introduce into the cells a sequence that directs the non-thesis of a ribozyme that then, specifically, will dissociate the necessary mRNA to synthesize the polypeptide whose expression is believed to be inhibited. The present inventors have found that the time of administration of the nucleic acid (in relation to the stage of the reproductive cycle) greatly affects the efficiency of the incorporation of the nucleic acid. The inventors have found that, in general, in order to obtain the optimum degree of incorporation of the administered nucleic acid, it is necessary that the administration be made in the period following ovulation, up to and including the day on which there is a maximum level of progesterone in the blood. The level of progesterone usually reaches its maximum at approximately a time similar to the point at which the embryo, if present in the uterus, can become implanted. Therefore, for example, the inventors have found that the maximum incorporation of the DNA administered by the mouse endometrium takes place on day 2-3 in the cycle (taken on day 1 as the day on which a vaginal plug is first detected). In humans, ovulation occurs, typically, on day 14 of the cycle, and implantation is estimated to take place, in general, in the central luteal phase (although the exact time is poorly defined in humans). The nucleic acid can be administered in pure form, or it can be linked or associated with other substances (for example liposomes). Conveniently, the nucleic acid is introduced into the cells of the mammalian container by simple transfection (with or without liposomes), which has been found by the inventors to be surprisingly effective, if the need for -sequence is introduced into a viral vector. Without being lazy, viral vectors may be convenient especially those which can be a reference point for certain types of cells (for example as described in WO 93/20221). The nucleic acid will be introduced, conveniently, as part of a construct (for example a plasmid, cosmid or the like), construction which, advantageously, will comprise an initiator, operable in a maniferous, for occasioning the transcription of at least part of the nucleic acid introduced. The initiator may be constitutive or more preferably inducible to allow greater ex-pressure control of the introduced sequence. In a particular method carried out according to the invention, the introduction of a molecule of nucleic acid into the endo-etrial cells of a female mammal allows the greater or lesser regulation of the vertility of the individual. The invention can be used, in particular, to provide a method of contraception for companion animals (e.g. cats and dogs) to avoid undesirable births. In other embodiments, the invention provides a method for improving the fertility of the species d? L. livestock such as pigs, cattle, sheep and the like.

Preferably, nuclucic acid is introduced into the reproductive tract through the vagina, which avoids the need for invasive surgical techniques. However, if necessary, the nucleic acid can be introduced by means of surgical techniques directly into the reproductive tract (e.g., inside the uterus). The invention offers the possibility of altering one or more of the features by introducing one or more than a very large number of different nucleic acid sequences. In one embodiment, the sequence introduced into the cells of the reproductive tract directs the expression (of pre-occurrence at high levels) of an effective portion of a cytokine or growth factor (an effective portion is that part of the molecule that retains the activity biologics particularly associated with the whole (for example, nothing to a specific ligand, etc.) Examples of these polypeptides that can be expressed by the introduced sequence include, but are not limited to, the following interleukins, lucemia inhibitory factor. (LIF), vascular endothelial growth factor (VEGF), epidemic growth factor 9EGF), heparin epidermal agglutinating growth factor (HBEGF), insulin agglutinating growth factors I and ?? (IGF-I YIGF-II), amfire regulin, colony stimulating factor (CSF), and factor for tumor necrosis (TNF). In another embodiment, the introduced sequence can direct the expression of an effective portion of a cytokine antagonist or a growth factor, such as the IL-1 receptor antagonist. Advantageously, the antagonist may be a soluble receptor for the cytokine or a growth factor. Suitable examples include the 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. In another embodiment, the induced sequence can direct the expression of an effective portion of a polypeptide having an immunological effect. In particular, the poly-peptide may possess immunogenic activity, thus serving to stimulate a local immune response, the invention can thus be used to provide a novel method of immunization. Advantageously, the immunogenic polypeptide will be an antigen of a mucosal pathogen. By virtue of the common mucosal immune system, the stimulation of antibody production in the reproductive tract can result in the production of the corresponding antibodies at distant rau-costal sites, such as the gastro-intestinal tract, the respiratory tract, lacrimal glands and similar. However, preferably, the antigen will be one of a pathogen that invades and / or colonizes the reproductive tract, typically a pathogen that causes a sexually transmitted disease. Examples include viruses such as IIIV, papilloma virus (for example HPV of various types), chlamydia and bacteria (for example N. gonorrhea). Alternatively, the polypeptide having an immunological effect can be an immunoglobulin or an effective portion thereof (such as a Fab, Fv, or scFv fragment, or an individual chain antibody). The immunoglobulin or an effective portion thereof can be directed against a pathogen such as a steroid? another hormone Thus, immunoglobulins or their fragments can be expressed locally to provide protection against disease or to regulate fertility. In another embodiment, the induced sequence can direct the expression of a polypeptide, or an effective portion thereof, which has an effect on menstruation. In another embodiment, the introduced nucleic acid can direct the expression, on the surface of the cells of the reproductive tract, of an effective portion of a receptor molecule. The receptor can be a receptor for a cytokine, a steroid hormone, or a growth factor (such as the EGF receptor, the TGFalpha receptor, or the VEGF receptor). A number of receptors that are described as "orphan" receptors are known, since the ligand that binds to the receptor is unknown. These receivers are of considerable interest in the pharmaceutical industry, since they can provide targets for novel therapeutic or prophylactic compounds. In accordance with the above, in another aspect, the invention provides a method for characterizing the biological properties of a polypeptide, comprising introducing the polypeptide-encoding sequence to be characterized within the cells of the reproductive tract of a mammal, and determining the effects of the expressed polypeptide. Preferably, the mammal is a laboratory animal such as a mouse or a rat. Conveniently, the polypeptide to be characterized will be an orphan receptor and, typically, at least part of its characterization will comprise the identification of its ligand. In general, the method will comprise the analysis of histological sections taken from the laboratory mammal, and their processing by any of several standard techniques (ie, histochemical staining, in situ hybridization, unological spotting, etc.). ). Therefore, the present invention offers a novel alternative to the steroidal regulation of endometrial function (and thus, reproductive capacity or fertility) by transfer of the gene in vivo. To achieve this, genetic engineering will be designed to specifically modulate the action of the cytokine. This can be achieved in a variety of ways. For example, cells that produce a secreted cytokine can be prevented from synthesizing the factor by blocking transcription and translation using antisense promoter or ribozyme promoters. Alternatively, the action of the secreted factor can be blocked by the receptor antagonist. Soluble receptors that take place naturally can sweep and neutralize the bioactive ligand thus acting as a competitive receptor antagonist. As an alternative, there are natural receptor antagonists, for example IL1Ra (antagonist re-ceptor interleukin-1). The intraperitoneal administration of this protein blocks the implantation of the blastocyst in raton (Simon and others, 1994 cited elsewhere). There is considerable evidence to show that the soluble growth factors secreted by the oviduct and the uterine epithelium can control the development of the pre-implantation of the embryo of the mammal, acting directly through the receptors expressed on the embryo (Pa pfer and others , 1990 IN Vitro Cellular and Developmental Biology 26_, 944-948). In turn, the development of embryos produces growth factors that can act in an antocrine manner, or on the endometrium to influence their receptivity. For example, in mice, LIF expression (from maternal tissue) is dramatically upregulated in the glandular epithelium on day 4, immediately before implantation. LIF is able to act on the pre-implantation of the - - blast cells, which express the LIF receptor (LIF-R). This maternal expression of LIF is vital for implantation since in mice destroyed with LIF, embryos will not implant, even though they will be transferred to pseudoembaraded mothers (Stewart et al., 1992 cited). The inventors have now extended this work to humans, and showed by RT-PCR, that human embryos express the mRNA that encodes LIF-R, but do not themselves express LIF. Llf acts by binding to a low affinity receptor LIF-R. The high affinity binding occurs when the LIF / LIF-R complex interacts with the accessory protein that translates the signal, gp130. Human embryos also contain mRNA that encodes this protein (Sharkey et al., 1995 Biology of Reproduction 55, 955-962). The inventors have also shown that the secretion of LIF in the glandular epithelium of humans is regulated by steroids, being maximum in the leuteal phase (approximately the expected implantation time - Charnock-Jones et al., 1994 cited elsewhere). In addition, the 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 mice. Clearly, cytokines can 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 disorganize or improve this communication, leading to novel methods of contraception or, on the contrary, improved implantation. The most current studies of the regulation of paracrine and autocrine reproductive function are limited to a descriptive analysis due to 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 vitro is feasible. This allows the endometrium to be implanted experimentally and offers new therapeutic strategies. The work outlined above describes the use of a reporter gene to demonstrate the practicability of uterine gene transfer in vivo. In practice, a gene (or other DNA construct) capable of altering uterine function will be used. Examples of these include receptor antagonists (eg, Il-1Ra, soluble CEGF receptors, etc.), natural or modified cytokines and growth factors, protease inhibitors or steroid receptors and a variety of ribozyme and antisense constructs. This work shows that the gene 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 humans, destroying the implantation (ie, contraceptives) endometriosis and menorrhagia , hyperplasia and adenocarcinomas. Using the protocols we have developed, the results described below were obtained. They demonstrate that gene constructs can be transferred to the endometrium both in vivo (in mice) and in vitro, and that these constructions are transcriptionally (and translationally) active. The invention will now be described, further, by way of illustrative example and with reference to the accompanying drawings, in which: Figures 1A and 1B show photomicrographs of histological sections of the mouse endometrium. transfected with (A) a plasmid construct that directs the expression of a reporter gene beta beta-galactosidase or (B) a similar plasmid lacking reporter gene. Transfected cells can clearly be identified by intense dark spotting (azu.1.) Within the cytoplasm, which is absent in section B; Figure 2 is a photomicrograph of a human endometrium in vitro, ie, of the cells of a human endometrium in vivo, which have been transformed with the same plasmid of Figure 1A - the dark (blue) staining due to the expression of the reporter gene is mainly associated with the remaining glandular structure, while the surrounding cells stained more weakly; Figure 3 is a bar graph showing the results of the CAT assay (in counts per tube) p < -? r_? -the endometrial cells successfully transfected :. with a gene coding for chloramphenicol acetyl-ansferase (pcDNA3CAT) compared to cells transfected with a control plasmid (pcDNA3), and Figure 4 is a bar graph showing the results of a luciferase assay (in relation to light proteins). ) for endometrial cells: 5 successfully transfected with a gene encoding lucifer .-- is .-- i (pcDNA3 LUC) compared to cells transfected with a control plasmid (pcDNA3).

E j e m p l o s E j us 1 Mice 3ALB / cJ NUlliparjus mature mice were housed in a light (light 14h, dark 10h, illuminated at 20.00h) and tem perature (22 ° C) Small Animal House controlled and fed a diet of mouse and rat ( Labsure, Christopher Hill Grup, Poole, Dorset, UK). They were placed with vasoctomized males of the same breed during the night and were examined the following morning for the presence of a vaginal plug. The plan was assumed to take place at 0.200h, time 0 and day 1 was counted as the day on which the plug was first detected.

Paired females were housed individually prior to experimentation. The laparotomy was carried out using aseptic procedures under metaf-anesthesia (methoxyflurane, C-Vet Ltd., Bury St. Edmunds). The uterine horns were exposed by central incisions. Injections were made either at the tip of the horn at the uterine tube junction or at the base of the horn at the uterus-cervical junction. Repeated studies showed that this last technique provides the best method of administration, but for some purposes the first was preferable when it was necessary to minimize disturbance of the reproductive tract. Liposome / DNA injections (pcDNA3 gene reporter beta-galactosidase), pure DNA or control solutions (25-100 mclt) were carried out by inserting the tip of a flat Stratatip into the base of the horn. Solutions were previously extracted within the tip by means of. A Travesty applicator that had also joined to control the slow injection of solutions into the horn. After the injection, the incision was closed using interrupted mattress sutures and the mice were allowed to recover in their cages and were provided with food and water. ad libitum Plasmid constructs The plasmids described in this application (as an example) are made in the commercially available pcDNA3 vector (Catalog No. V790-20, from Invitrogen, San Diego, Cal., USA). Plasmid PCDWA, without any reporter gene, was used as a negative control. The experimental plasmids pcDMA3-betagal, pcDMA3-CAT, and pCDNA3-Luc contained beta-galactosidase, chloroamfeicol acetyl transferase and luciferase, reporter genes, respectively. These plasmids contained the following genetic elements: Ampicillin-resistant gene, original replication ColEl, CMV initiator, [reporter gene], bovine growth hormone polyA additive, fl origin of replicate, SV40 replication origin , neo-icine-resistant gene and an SV40 polyA ad-tion site, in operable relationship such that the reporter gene can be expressed in eukaryotic cells after the introduction of the plasmid. The DNA plasmid was purified from E. coli by alkaline lysis and further purified using Qiagen ion exchange column (according to the manufacturer's instructions).

Preparation of Liposouvi The liposome used was a J: 1 (w / w) lipid formalization of DOPSA. { '(sperminecarboxamido) effile] NN-diptethyl-1-p.ropanamine) and DO.VE (dioleylphosphatidyl ethanolamine). (LipofectAMINE; Gibeo BRL Paisley, Ssotland) A number of DNA, lipid proportions - and Different injection volumes were used as shown in Table 1. Immediately DNA / iiposomes were mixed in each experiment, 10 mclt of a DNA solution was added to 10 mclt of lipid solution, mixed moderately and leaving for 15 minutes at room temperature, 80 mcL of PBS was then added to give the final DNA and lipid cone, as shown in Table 1. This was injected into the uterus of the pseudoem-embraced mice (see previous section). for details of the mice and surgery).

Histochemical localization of beta-Galacfcosidase The animals were killed by inhalation of carbon dioxide and the horns of the uterus free of fat and mesonomyelitis were dissected. Each horn was divided into 3 sections with the upper section and the lower one being frozen ", in liquid nitrogen, and stored at -70 ° C before the counting of the car gone from bet-ga aatosida a. After the heat was fixed to the glutpraldts at 1.25% in P3 'ur.H t' 15 minutes, it was rinsed in P74S times and placed in sun. coa X-gal (1 / ng / ml gal. 5 aM K3Fe {CN) g,. "? TIM MJCI ,, 0.02% NP40, and 0.01% sodium deoxychola (for 24 hours at ambient temperature. The sections were then rinsed in PBS / 3 '. DMSO (2 x 5 minutes), 70% ethanol (3 x 5 minutes) and placed in 100% etaaol - The tissues were embedded in either metatoryl or glycol and cut 7-membered scissors and arouse neutral red blood before microscopic examination.

Results Table 1 shows the various conditions used for the intensity of staining resulting from the uterine sections after administration of the DNA and liposome. The results shown in Table 1 show the critical state of the regulation of the time of the administration of the plasmid DNA, giving the improved levels of expression on day 2 administration, giving reasonable levels an administration on day 3, but the administration on day 4 it resulted in very little expression of the reporter gene, I presumed lamente because the endomstrial cells do not to marón the construction in í > This point of time, for reasons that are not completely clear.

/// TA3T.A nía of DNA Lipid Volume of Intensity alministr. spot injection (day of (mcg / ml) (mcg / ml) do histoquí autopsia) (mclt) mico 2 (5) 2 20 50 + -r 3 (5) 2 20 50 ++ 4 (6) 2 20 50 2 (6) 2 20 50 ++ ¡- 2 (5) 8 10 20 + f 44 ((66 )) 88 2200 50 4 (6? 30 0 50 4: 6) 2 60 50 laten ?, "stain-likeness ++" - force, ++ moderate, + weak, nlnjn Control - A: a.: '"N? Ssudo" span of 5 1 as: not injected > did not give a uterine signal for beta-activity: j-ilact < "» 3idana, A mouse.-, pseudoemb ~: reasoned (administration day 2, autopsy day 6) injected with 50 mclt of pDNA3 minus beta-galactosidase (2 mcg / ml) and lipid (20 mcg / ml) did not give uterine staining For the beta-galaotosidase activity, the examination of the histological sections after staining with X-gal showed that the glandular epithelium was strongly stained and the luminal epithelium was also stained but less strongly.The optimal spotting was observed in animals trinsfeot two. with 2 mcg / ml of DNA and 20 mcg / ml of lipid in 50 mclt admired on day 2 of the psuedoemb razo, the figure la our a section of this animal, and figure lb shows a section of a control animal when rs received (under identical conditions) a plasmid that lacked the beta-gal gene.

Example 2 Transfection of primary cultures of human andometrium. The inventors have also demonstrated that the human endomatrial epithelial cells can be refined in vitro at high efficiency. The same plasmid was used: 'pcDN? 3, +/- beta-galac-tosidase gene reporter) and the lipids already described. The endometrial cells were prepared by the Sm.lth method.

Kelly (Smith et al., 1987 Pr st glandins 34. '553-561). Once the cultures were established, the following transfection protocol was used. Each »AON (2 mcg) and liposomci (8 mcg) were diluted in 130 raclt of a serum free medium (Optl-MEMl 3RL), mixed and incubated at room temperature for 15 minutes. After this, 800 mclt of Opti-MEMl was added. Cells (in 24-well plates) were washed with PBS followed by washing with Opti-MüMl. The mixture of? DN / liposome (0.5 ml) was then added to the cells and incubated at 37 ° C for 3 hours in an incubator of - C0_ after which 0.5 was added to the culture medium containing 20% fetal calf serum. The cells were fixed: 0.1%, rinsed and stained with X-gal 24 oras after transfection. This work showed that the genes can be trpns faridos to endo < In vivo and this will find use in many endometrial (and piaoential) conditions, for example, improve implant ion in both animals in the omen, destroy implantation (ie, contraception), eadoiaetriosi? and nienorr'igLa, hyperplasia and edenocarclnoma. Additional data were obtained that are related to the in vivo trinfection of human, purified uterine epithelial cells. This complements the work of the rjón in vivo shows that similar cells / after a minimum time in the culture, can be efficiently transfected with the same liopo ome and the used DNA inivo, Example 2 Transfection of epithelium in lometria! human in vi v > Rimary epithelial cells; The cells of the endometrium were isolated and cultured according to the method of I'.hang et al., (Cell 3ci «nce, 1935; 108: 323-331). receptacles, standard to achieve: a density of 50% confluence the next day.The cells were cultured for 5 days, then coated again with plates of 24 receptacles, at a density of 60,000 cells per-receptacle. Next, the cells were transfected with DNA / liposomal complexes (DNA / LC), which were prepared as follows: Procedure of Trafection LipofectAMINK apparatus (Gimeo Catalog No. 18324--0L2), Opti-MHMi (Gibco Catalog No. 51185-018). 24 receptacle culture dish The cultivation dish was as described by Zhang et al. (Cited above). Estft consists of DMEM / HE ES, 10% FCS, Endofcheliai-the Growth Supplement (Sigma Catalog? To, E-2'759), at 30 mcg / ml heparin (Sigma Catalog Mo, H-3149), at 90 mcg / ml, IgA Aigin gene Catalog No. G-1272), at 5 μg / l, and fungi-zone (Gioco Catalog No, 15290-01) at mcg / ml. Mg + r, free Ca-V + PBS and an eppendorf tube of 2 raiiiliters were also used. Different plasmid constructs containing different reporter genes were used. 3 obtained pcA N3CA.T from Invitrogen Corporation, and contained a reporter gene that encodes the ima. chlor. > nmfenicol acetil tran_¿ ferasa. The second plasmid p ^ W-T ': ^ comprised the same vector, but the CAT gene was recombined with the gene coding for the firefly luoiferae. Large-scale DNA preparations of the vectors were made using the Qiagen midipr ^ p system. As a negative control; pcADNS was used that did not contain a reporter gene, Preparation of AD / Liposoa complexes 1) Solution A A DNA nicrogram diluted in 100 raclt of OpiMEM in an Eppendorf tube. DNA was used at a final concentration of 1 mcg / ml in the transfection medium. * 2) Solusion B Four miRograms of Li? OtectAM :: NF di Luido in 100 mclt of Opti-MUMI w.-? an Eppendorf tube. Lipof ct-MINli was used at a final concentration of 8 mcg / ml in the transfection medium. 3) Combine two solutions A and B in a new tube and mix moderately 4.}. Hide the te¡nper.??: Ur.-? environment for 5 minutes.

Rinse the Cell 1) Before the treatment, e-rinse the cells of monkey cap ?, approximate - 3 times in PBS F .5S without serum. 2) Re-rinse the two-layer monolayer cells with Opti-MKM 1.

Trans eccon 1) _ Add 300 mclt í t t =? L 1 .0 ml) of Opti-MEM to each tube that cont-; 'ozcla of DNA / lipid. Final concentration of DNA 1 mcg / ml, and concentration of lipofect- AMINE 8 mcg / ml. 2) ftetaover the Opti-MEM i in the monolayer of the cell. 3) Gently mix the DNA / LC mixture and superimpose the diluted complex solution on the washed cells, 0.5 ml / receptacle 24. 4) Incubate the cell monolayer for 3 hours at 37 C in a C0_ incubator.

Additional Culture of Cells 1) Three hours later, the transduction mixture was re-aerated and 2 ml of Zhang's medium was added to each receptacle, and it was grown adi.tionally.

Test Cuant lt =? T ivo From 24 to 43 hours after trpnsfection, the cells were extracted and stained for CAT activity or luciferase reporter. Sagún was appropriate. 1. Rinse the cells three times in PBS. 2. Extract the cells with 300 microliters of Lysis buffer (Promega Catalog No. E-3871), thoroughly scraping the cells thoroughly with Eppendorf. 3. Freeze the extract quickly at -70 ° C and reserve until the test. 4. To test, the extracts were thawed and centrifuged at 13,000 g for 5 minutes. 5. Repeat the freeze / thaw cycle to see one more time. 6, Report the activity of the gene repórt -? - i .- * lucíferas. * Using the equipment d * ¿- trial Lüciferas-t Troplx (Catalog no, BC100L), 40 riclt of each extract per tube was used, 7 The activity of "1 gene repórter CAT was tested using the equipment Quan-t -CA ': 1 of? Mersha:! (Catalog No. TRK 1012), Results The primary endometrial epithelial cells were transfected into plates of 24 receptacles as described above. Trpnsfections were carried out on triplicate receptacles with pcDNA3 (as control), pcA NCA, and pc DN3LUC. After 48 hours the cells were harvested and assayed for luciferase or CAT activity. The CAT ion catalyzes the transfer of the acetyl groups from the very low A of acetyl to the cioramf nicol. The use of tritiated acetyl coA results in the transference of radiolabel to chloroamfenicol. The CAJ * activity in a sample is directly proportional to the amount of tritiated chloramphenicol produced. Therefore, the results are expressed in pm per tube. A conventional curve can be produced by roasting lysis buffer that contains known amounts of purified CAT. The results are shown in Figure 3, which is a bar graph showing the + sem for triplicate determinations for a typical experiment. The results in numerical form were as shown below, with the CAT activity in the cells treated with pcDNA3CAT approximately 6 times higher than the control samples, demonstrating an exit use transfection of the endometriotic cells. PCA0N3 pcADN3CAT 710 4480 615 4134 662 3234 medium 662 + 271 3951 + 372 In the pair * luciferase assay, the cell extract containing luciferase was mixed with its luciferin substrate .. resulting in the emission of light, The intensity of the light signal is proportional to the luciferase enzyme present in the extract, and can be measured by means of a luninometer. Initial results are given in units relative to light.

The results sa show in Figure 4 that it is a bar graph illustrating whether madio _sem for triplicate determinations for typical experiments. The results in numerical form were as shown below. The signal from the cells treated with pcDNA3 UC were more than 30 times higher than the background signal from the controls, demonstrating again the successful transfection of the endometriotic cells.

Examples of possible applications of endometrial gene transfer At least seven different types or constructs of genes could be transfected within the endometrium to achieve a variety of different effects. Each of these different types will be described in turn. 1) Over-expression of cytokines and growth factors. These are conceptually the simplest types of constructions in which they will be designed to over-express either a cytokine or a growth factor in the uterine epithelial cells.Examples of CADN suitable for this • over-expression, include those They codify IF, VEHF, EGF, CSF, TN. '', Amfiregulin and a variety of interleukins and colony stimulating factors.These have been moatra "which are naturally expressed in the endometrium and are thought to be important in the role of referencing in the trio, (Stewart et al., 1992, Nature, 359, 76--70; Char.uook-Jones et al., 1994, Journal of Reproduction, 48, 1120-1123; Das ot s, 1995, Molecular E docribology, _9, 691; Tab.i'o-: adeh (1994, HumanReproduc ion Update, - 9_, 947-957) nan publish an extensive review in this field). These agents affect different ats of reproductive function, including implantation, blood vessel development and leukocyte biology. Therefore, possible indications for the administration of these constructions will be able to be used to improve the ethos, particularly in cattle, or to avoid the conception of humans and their accompanying animals, and also to treat an emptiness of disorders. -nstrual s in humans * i. ') An example gives a designated example to improve fertility in livestock It is h =. The demonstration of T.TF is essential for the implantation (ñtewart et al., 1992 cited above). It is • 0 factor is produced by him endometrium ar! , -mment of implantation. Therefore, it is possible that in spices where arabic ion loss regimes are high, the increase in LIF expression levels of the endometrium at the time of implantation can reduce these proportions. gives loss. Thus; The transfection of a gan designed to direct the LIF syndromes of the endometrium at the time of implantation can improve fertility in these species. Constructs trans- mitted within the endometrium may need to contain regulatory sequences to ensure that the LTF protein was produced at the appropriate time. Is it likely that this could be achieved using the LI gene promoter? of the species in question. Treatments designed to relieve: a menstrual dysfunction in women can also be considered using the endometrium- gan transfer. A »> This can be the purpof altering the development of the blood vessel in the endometrium during the transfection of the gene encoding the angiogenic growth factors, for example VEGF. A high increase in VEGE production can be expected to improve capillary growth and, therefore, can promote endometrial thickening. Similarly, increased levels of VEGF can facilitate the repair of capillaries after menstruation and thus alter the bleeding patterns treated with this type of construction. 2) Sobr-: ~ e? Receptor resclón The increase in the number and type of receptors expressed by the uterine epithelium can be anticipated to have significant biological consequences. The types of receptors that one may wish to express include, but are not limited to, growth factor and cytokine receptors, for example EGF, TGFalpha, VEGF and a variety of colony and interleukin stimulating factors. The steroid hormone receptors are also suitable for expression in epithelial cells. One might expect this transfection to be useful where one wishes to improve fertility, avoid concision, treat menstrual disorders and also elucidate the function of orphan receptors (orphan receptors are the receptors where the ligand is not identified). Orphan receptors represent an area of great interest in the pharmaceutical industry, since the characterization of the ligand can lead to the generation of new drugs. It has become 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 through statins. In particular, it has been shown (Nelson et al., 1992, Endocrinology, 131, 1657-1644) that TGFalpha is a potential mediator of the action of estrogen in the uterus of the mouse. Therefore, the transfection of the constructs that direct the synthesis of this factor may encourage it to promote endometrial erection and, therefore, may increase the fertility in situations where the endometrium has not developed adequately. Similarly, this factor can be anticipated to promote repair of the epithelial surface after menstruation and, therefore, be useful in the treatment of hemorrhage. There are several members of the superfamily steroid hormone receptor for which the ligand is usually unknown. The transfection of each of the cDNAs within the endometrium can be of great benefit in enhancing the biological function of these receptors and, therefore, they can find application in the search for new pharmaceutical agents that act on these receptors. (Evans 1988, Science 740 »889-895. 3) Transfection of constructions designed to block or prevent the action of the growth factors of the cytokine and other hormones.

The transfection of the natural antagonists to the cytokine and growth factors open up the possibility of -nodulating the endometrial function. An example of these antagonists may be the interleukin 1 receptor (Hannum et al., 1990 Nature 343, 356). The administration of this protein has been found to block pregnancy in mice (Simon et al., 1994 Endocrinology 134, 521-528). Other natural antagonists of cytokines and growth factors include natural soluble receptors. Soluble re-captors have been described in a variety of growth factor / cytokine systems. For example, TNF (En-gelmann et al., 1990, J. Biol. Chem. 265, 14497-14504), FGF (Gi ol 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, 1490-1414). The common feature is that the binding domain of the extracellular ligand of the receptor is released from the cell as a freely soluble factor. This is achieved either by proteolysis or by alternating slip that generates a truncated protein, that is, it generates a truncated protein molecule lacking the transmembrane and the intracellular domains. Kendall and / Thomas (1993), Proc. Nati Acad. Sci, EüA, 90 »10705-10709) describe a soluble variant of the VEGF receptor. This protein was able to block the action of VEGF in vitro. We have isolated three additional cDNAs that encode additional soluble variants (see PCT / GB95 01213). The use of these natural agents has several advantages over other antagonists (for example anti-CEGF antibodies). Since it takes place naturally in the body, it could be anticipated that they can not elicit an immune response and must be well tolerated. Also, since they are membrane-bound receptor derivatives, the binding characteristics will be very similar and thus, they will compete very effectively for the ligand. It is possible that there are other receptors naturally soluble or that can be worked in vitro. It is likewise probable that if the ligand-binding domain forms a member for the steroid hormone receptor it was expressed it could act as a dominant negative receptor in which it can compete for the ligand if it is expressed within the cell at sufficiently high levels . As an alternative, a non-activating "receptor" can be used, but one that binds the DNA to block the transcription of the gene. This application may be useful for antagonizing action on natural steroids including those which are ligands not yet identified for orphan receptors (Pamricks et al., 1994 Leu eia 8, 1797-1806). Pointing deficient receptors of the seven family of transmembrane domain receptors can also be worked on and transferred. The soluble interleukin-1 receptor agonist (Eisenberg et al., 1990 Nature 343, 341), has been shown to antagonize the actions of IL-1 in vivo (Simón et al., 1994 Endocrinology 134, 521-528). Thus, the transfection of the endometrium with a cDNA construct intended to direct the synthesis of the antagonist can be expected to block pregnancy in mice. Another likely factor for antagonizing the growth factor or action of the cytokine includes the soluble variants of the natural receptors, for example the soluble variant of the VEGF receptor has been described by Kendall & Thomas < 1993 > , above cited > and also by Boocook and others. < 1995 J. Nati. Caoncer Ins. 7, 506-516 > . Local production of other factors can be expected to antagonize the actions of VEGF and may lead to useful therapeutic use in situations where there is hyperproliferation of epithelial cells, for example, in a variety of menstrual disorders where it is desirable to reduce capillary density in the endometrium. This can include a malignant disease. 4) Use of antisense methods to avoid the local oiox o of a specific protein (or enzyme) An alternative approach to block action: "Cytokines, growth factors and hormones may be the use of ribozyme technology to block either the production of ligands or the production of receptors in appropriate cells (James , 1991 Antiviral Chemistry and Cheraotherapy, 191-214, Albert &Morris, 1994 Trends in Pharmaceutical Sciences 15_r 250-254 Antisense technology has the binding of the so-called antisense oligonucleotide or polynucleotide for a cellular mRNA. the translation of this mRNA and, therefore, reduces the amount of appropriate protein produced by the cell.The synthetic oligonucleotides or polyribonucleotides have both been successfully used for this purpose.The measured transfection of the oligonucleotides liposome or mediated transfection of the 1iposorna of the DNA constructs that direct the synthesis of poly-ribonucleotides can be expected selectively reduce the protein reduction by means of the transfected cells. The ribozymes also prevent production by selective dissociation of RNA encoding the specific protein in question. These can also be transfected as polyribonucleotides (for reviews see James 1991, and Albert &Morris 1994, both cited above). One example of this use of an antisense ribozyme to prevent fertility may be as follows. It has already been shown that LIF is essential for the implantation process of mammalian engraftment (Stewart et al., 1992, previously cited). Therefore, a transfection of any oligonucleotide or DNA constructs that direct the synthesis of polyribonucleotides. Antisense, or robozymes directed against the LIF mRNA, can be expected to avoid the syn- thesis of this factor. The lack of this factor must then lead to a failure in the implantation and, therefore, the conception can be blocked. A similar approach can be used to block the production of angiogenic growth factors, for example VEGF, which can prevent the proliferation of ando-telial cells required for the growth of a tumor. Therefore, this type of therapy can be particularly advantageous where a malignant disease is being treated.

) Local production of immunoglobulin and its fragments It is possible to use the technology of. Modern recombinant AON for generating antibodies from a single strand that has? S-fi characteristics almost identical to the whole-moaoclonal antibody from which they are derived. These indivudal chain antibodies have been successfully expressed in bacteria (He et al., 1995 Immunology j4, 662-668). It is possible in principle to work a construct that will direct the expression of an individual chain antibody and express this in the epithelial cells. If you have taken alcabo in the endometrium in vivo, it can be anticipated that the single-chain antibodies directed against a steroid hormone will bind to the steroid and prevent its action in the ergotemporal cells. An exemplary of this antibody may be the single chain antibody derived from the mono-clonal antiprogesterone DB3 (He et al., 1995, cited above). If this antibody were secreted within the uterine lumen, it could also bind progesterone and may have actions in the uterine compartment. The antibody directed against the growth factors and the cytokines that are known to activate the endometrium can similarly block its function if it is produced locally in this way. An additional application for the locally produced individual antigen antibodies may be to prevent or treat sexually transmitted diseases. In this location, the antibodies directed against the gene in question (eg, papillone virus, HIV, chlamydia) can be secreted into the uterine lumen and avoid infection by the agent in question. Antibodies directed against the perma or oocyte antigens can be considered to play a role in contraception. 6) Active immunization to achieve mucosal immunity An additional method that can be used to achieve local immunity may be to design constructs that can direct the secretion of an antigen into the lumen. This can elicit a local immune response and thus can achieve the site of specific mucosal immunity. It has been known for many years (Howe, 1967 Journal of Reproductive and Fértility 1 ^ 3, 563-566) that the uterine lumen contains many leukocytes. It is possible that the antigen produced by transfected endometrial cells taken by these leukocytes and then presented to elicit a mucosal immune response. The administration of the antigens to the intestinal lumen has resulted in this immunity and, in some cases, has been shown to be very effective (for example vaccination against poliomyelitis). 7) Sites to bind the blocking pathogen The binding of pathogens to the uco-salts surface is frequently an essential requirement for the establishment of the infection. The blocking union of pathogens at these sites can thus present a method of protecting humans or animals against diseases, particularly sexually transmitted diseases. This applies not only to bacterial pathogens (such as certain pathogenic strains of E. coli and N. gonorrhea) but to viral pathogens. Many viruses when they infect a cell are joined by means of a "receptor" on the surface of the cell. The local production of soluble receptors can be expected to compete with the molecules on the surface of the cell and thus avoid viral infection. Likewise, the saturation of receptors on the surface of the cell with viral mimics (which act as the viral "ligand") can also block infection, such as the local production of specific Imaunoglobulins or their effective binding portions.

Claims (26)

R E I V I N D I C A C I O N S

1. A method for altering one or more characteristics of at least some of the cells of the reproductive tract of a female mammal by introducing a nucleic acid into the cells.

2. Method according to claim 1, wherein the female mammal is a human.

3. Method according to claim 1 or 2, wherein the nucleic acid is introduced into the endometrial cells.

4. Method according to any of claims 1, 2 or 3, wherein the nucleic acid is introduced into the glandular epithelium of the endometrium.

5. Method according to any of the preceding claims, wherein the introduced nucleic acid is DNA.

6. Method according to any of the preceding claims, wherein the nucleic acid is introduced in the period following ovulation, up to and including the point at which there is a peak in the level of progesterone in the blood.

7. - Method according to any of the preceding claims, wherein the nucleic acid is introduced into a liposome, a virus or other carrier particle.

8. Method according to any of the preceding claims, wherein the nucleic acid is introduced as a plasmid or other construction.

9. Method according to any of the preceding claims, wherein the introduced nucleic acid comprises a promoter operably linked to a sequence to be transcribed in a eukaryotic cell.

10. Method according to claim 9, wherein the promoter is inducible.

11. Method according to any of the preceding claims, wherein the introduced nucleic acid sequence comprises a portion operably linked in antisense orientation to a promoter, with the o? In of exhibiting the expression of a polypeptide in the cells within the which the sequence is introduced.

12. Method according to any of the preceding claims, wherein the characteristic altered by the introduction of the nucleic acid sequence results in an alteration of the fertility of the entity.

13. Method according to any 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.

14. Method according to any of the preceding claims, wherein the introduced nucleic acid directs the expression of at least an effective portion of one of the following: an intereucine; a leukemia inhibitory factor (LIF); a vascular endothelial development factor (VEGF); an epidermal development factor (EGF); an epidermal heparin binding development factor (HBEGF); factors I and II for the development of insulin binding (IGF-I and IGF-II); Amphiregulin; colony stimulation factor (CSF); tumor necrosis factor (TNF); hepatocyte development factor (HGF); and fibroblast development factor (FGF).

15. Method according to any 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.

16. Method according to claim 5, 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) a; fibroblast development factor (FGF); factor of development of derived platelets (PDGF); interleukin-6; vascular endothelial development factor (VBEGF); or a hepatocyte development factor ("Met" receptor).

17. - Method according to any of claims 1 to 12, wherein the introduced nucleic acid directs the expression in and / or the cell of at least an effective portion of a receptor for a developmental factor; a cytokine or a steroid hormone.

18. The method according to claim 17, wherein the introduced nucleic acid sequence directs the expression of at least an effective portion of a receptor for one of the following: EGF; Transformation development factor (TGF) a; or VEGF.

19. Method according to any of claims 1 to 12, wherein the introduced nucleic acid directs the expression of at least an effective portion of a polypeptide having a local immunological effect.

20. Method according to claim 19, wherein the introduced nucleic acid directs the expression of at least an effective portion of an antigen of a pathogen.

21. The method according to claim 19 or 20, wherein the introduced nucleic acid directs the expression of at least an immunogenic portion of a polypeptide of one of the following: HIV; papilloma virus, Chlamydia or N.gonorrhoea.

22. Method according to claim 19, wherein the introduced nucleic acid directs the expression of an immunoglobulin or an effective portion thereof.

23. A composition comprising a nucleic acid for use in the method of any of the preceding clauses.

24. Use of a composition comprising nucleic acid, according to any of clauses 1 to 22.

25. - 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 female mammal. 26.- Method for preparing a composition for use in the method of any of claims 1 to 22, this method comprises mixing a nucleic acid with a physiologically acceptable carrier substance. SUMMARY A method for altering one or more characteristics of at least some of the cells of the reproductive tract of a female mammal is described by introducing a nucleic acid into the cells, together with a composition comprising nucleic acid for use in the method.