WO2005039282A1 - Drosophila transgenic animal models for the treatment of human genetic diseases caused by expansions of microsatellites which contain the trinucleotide ctg - Google Patents

Abstract

The invention relates to Drosophila transgenic animal models used to treat pathologies wherein the presence of expansions of microsatellites based on the sequence CTG has been detected, such as myotonic dystrophy of type 1 and 2, the spino-cerebellar ataxy 8 and HSL2. The invention concerns the application to a transgenic animal model of a transgene which includes totally or partially a microsatellite sequence based on a sequence CTG. This transgenic model has been obtained through the stable integration of the transgene into the melanogaster Drosophila genome. In said animals the transgene is expressed in a phenotypical and controlled manner at any of its development phases. The animal models of the invention apply to the investigation of pathogenesis mechanisms in diseases wherein the pathological states show expansions of microsatellites containing the sequence CTG, as assay devices for possible therapeutical compounds for the treatment of said diseases and as devices for the in vivo validation of potential treatments of those diseases.

Description

 DROSOPHILA TRANSGENIC ANIMAL MODELS FOR HUMAN GENETIC DISEASES CAUSED BY EXPANSIONS OF MICROSATELITES CONTAINING THE CTG TRINUCLEOTIDE

Technical sector This invention has utility in the bio-edicine sector and in the biopharmaceutical sector. Within these sectors, the specific field of utility is the use of transgenic animals as models that allow the investigation of hereditary human diseases, the development of treatments against them and the validation of potential pharmacological treatments.

State of the art The aberrant expansions of micro-satellite sequences in the human genome, understood as repetitions of a sequence of between two and seven nucleotides, are the origin of a growing number of inherited diseases. These include myotonic dystrophies (DM), a type of frequent dystrophy in adults, ataxias, especially spinocerebellar ataxia 8

(SCA8), and a disease very similar to the disease of

Huntington (HDL2 or "Huntington disease-li e 2") all due to the expansion of a microsatellite containing the CTG sequence. Myotonic dystrophies constitute a group of autosomal dominant multisisystemic diseases that are characterized mainly by myotonia (inability to relax muscles) and myopathy (muscle degeneration). Ataxias are a complex group of neurodegenerative diseases that lead to generalized uncoordination of limbs, gait and speech. Molecular studies have identified several microsatellite expansions as responsible for these diseases. Specifically of the CTG trinucleotide in the 3 'untranslated region of the DMPK gene (type 1 DM) or in a untranslated region of the Sca8 transcript (SCA8), or the CCTG tetranucleotide in the first intron of the ZNF9 gene (type 2 DM). In the case of HDL2, the situation is less clear, but it is known that by alternative maturation of the primary transcripts of the junctophyllin-3 gene, transcripts containing between 51 and 57 repetitions of the CTG triplet in intron 1 or in 3 'region not translated. Recent studies conducted in vitro and in vivo on biopses of patients affected by DM (Miller et al., 2000; Mankodi et al. 2001 / Fardaei et al. 2002) have shown that expansions associated with myotonic dystrophies bind to proteins homologous to the Muscleblind (Mbl) protein of the Drosophila melanogaster insect. This finding has led us to propose the following pathogenesis model: the expansions sequester all, or part, of the Mbl family proteins found in the nucleus of human cells and this sequestration prevents Mbl proteins from carrying out their function. normal physiological (not yet clarified). Thus, it would be the absence of free Mbl proteins responsible for the onset of pathogenesis in patients affected by myotonic dystrophy. This idea is supported by the fact that mutant flies for the muscleblind gene show defects similar to those presented by patients affected by myotonic dystrophy, such as the absence of a structure of the sarcomeres (the molecular motors of the muscles ) called disk Z. Similarly, the mutant larvae of Drosophila have a hypercontraction of the muscles, which is homologous to the myotonia shown by patients affected by myotonic dystrophies (Artero et al., 1998). However, other pathogenesis routes that probably contribute to the clinical manifestation of these diseases have also been proposed.

'medades. Thus, it has been proposed that the expansion of CTG microsatellites in the DMPK gene could lead to reduction in DMPK protein (DMPK haploin-sufficiency hypothesis) and that the microsatellite could have a local effect on chromatin structure leading to a reduction in the expression of nearby genes and / or an alteration in the acting domains of nearby transcription enhancing elements (hypothesis of the local effect on chromatin). At this time no specific pharmacological treatment is available for patients affected by any of the myotonic dystrophies or by SCA8. The only treatments available are aimed at relieving patients' symptoms. However, studies on the pathogenesis mechanism of microsatellite expansions that include the CTG sequence open the door to find possible therapeutic targets. The critical steps in the identification and development of new therapeutic agents are: (a) generating the candidate therapeutic agents; and (b) the systematic search among the candidate compounds of those that are safe and effective. With the advent of methodologies based on combinatorial chemistry, large quantities of potentially therapeutic compounds, known as libraries (collections) of compounds, can be generated. Likewise, based on the biological diversity presented by the Biosphere, there are collections of plant extracts, marine animals, etc. These collections constitute ordered sets of potential therapeutic agents. After generating the potentially therapeutic compounds, these promising candidates should be systematically searched for, that is, with a desired biological activity. Protocols for systematic searches of high performance in vitro have been developed. However, these trials for systematic searches must be complemented with in vivo tests. Since it is not ethically It is acceptable to test directly on compounds with a potential therapeutic activity, an important step in the identification of compounds with a desirable biological activity in humans is their prior use in systematic searches in non-human animal models. As such, non-human animal models of inherited diseases such as myotonic dystrophies and SCA8 can play an important role in the discovery of therapeutic agents to combat such diseases. A complementary strategy in the search for compounds with a desirable biological activity is to elucidate the pathogenesis molecular pathway of the disease. That is, to establish the cause-effect relationships that lead to the development of the symptoms of that pathology. The more elements of the pathogenesis pathway identified and their cause-effect relationships established, the more possibilities will exist to intervene in a predictable manner in the correction of the molecular defect. An adequate strategy to elucidate the pathogenesis pathway of a disease (which also does not require any prior knowledge of its molecular mechanism) is genetic analysis. One type of non-human model animal that can be used for systematic searches of therapeutic agents for the treatment of hereditary diseases and their genetic analysis are animal models in non-human mammals, for example the mouse. However, mice are expensive to maintain, have a slow reproduction time and produce reduced offspring. Thus, the mouse is not an ideal organism for high-performance systematic searches or for genetic analysis. For all the above, there is a need to describe additional model animals as a model of human diseases encompassed as caused by expansions of microsatellites containing the CTG sequence. Of particular interest would be an animal model with a short longevity, a rapid reproduction cycle and producing a large amount of offspring. Preferably, such an animal should be relatively simple and cheap to keep in the laboratory. Relevant patents for this application are: US Patent "Transposable elements as transformation vectors" with number 4,670,388 and US Patent "In vivo high throughput toxicology screening method" with number 6,365,129. Also relevant is the Spanish patent application with number P200201454 and priority date June 18, 2002 entitled "Transgenic animal models in Drosophila for myotonic dystrophies", in the name of this same applicant. The methods for the production of transgenic animals in Drosophila melanogaster are described in: Spradling, AC, and Rubin, GM (1982). Science 218, 341-347; Brand, AH and Perrimon, N (1993). Development 118, 401-415; Phelps and Brand (1998). Methods 14, 367-379; Steller and Pirrotta (1986). Mol. Cell Biol 6, 1640-1649. See also Spradling AC, P element mediated transformation in Drosophila: A practical approach (ed. DD Roberts, IRL Press, Oxford) (1986) pp. 175-179. The articles describing the muscleblind gene and its functions are: Artero, RD (1995). Molecular characterization of the 54A region of Drosophila melanogaster. Doctoral Thesis University of Valencia; Artero, R, Prokop, A, Paricio, N, Begemann, G, Pueyo, I, Mlodzik, M, Pérez-Alonso, M, Baylies, M (1998). Dev. Biol. 195, 131-143; Begemann, G, Paricio, N, Artero, R, Kiss, Pérez-Alonso, M and Mlodzik, M (1997). Development, 124, 4321-4331. García Casado, Z (2002). Functional characterization of the muscleblind gene: Drosophila assessment as a model for the study of Myotonic Dystrophy.

Doctoral thesis (in preparation). University of Valencia Articles describing the relationship between the molecular behavior of the expansions responsible for myotonic dystrophies and muscleblind proteins are: Fardaei M, Larkin K, Brook JD and Hamshere MG (2001).

Nucleic Acids Research 29, 2766-2771; Mankodi et al. (2001). Human Molecular Genetics 10, 2165-2170; Fardaei, M,

Rogers, MT, Thorpe, HM, Larkin, K, Hamshere, MG, Harper, PS and Brook, JD (2002). Human Molecular Genetics 11, 805-814;

Miller, J, Urbinati, CR, Teng-umnuay, P, Stenberg, MG,

Byrne, BJ, Thornton, A and S anson, MS (2000). The EMBO J.

19, 4439-4448. Articles describing microsatellites as responsible for human diseases and a possible function of microsatellites with CTG sequence: Ranun, LP and Day,

JW (2002). Current Opinion in Genetics & Development 12:

266-271 ^" ; Philips, AV, Timchenko, LT and Cooper, TA (1998).

Science 280: 737-741; Richards, RI (2001). Human Molecular Genetics 10: 2187-2194; Cummings, CJ, Zoghbi, HY (2000).

Annu Rev. Genomics Hum. Genet 1: 281-328; Filippova, GN et al. (2001). Nature Genetics 28: 335-343; Koob, MD et al.

(1999). Nature Genetics 21: 379-384. Articles describing other animal models for myotonic dystrophies are: Mankodi, A et al (2000). Science 289, 1769-1772; Seznec et al. (2001).

Human Molecular Genetics 10, 2717-2726. Articles in which the genetic technique of systematic search for genetic modifiers of a given phenotype is used are: Hays, TS et al. (1989). Molecular and

Cellular Biology 9, 875-84; Deuring, R, Robertson, B,

Prout, M and Fuller, M T (1989). Mol. Cell Biol. 9, 875-84;

Fuller, M T et al. (1989). Cell Mot. Cyto 14, 128-35; Y

Rottgen G, Wagner T, Hinz U (1998). Mol. Gen. Genet. 257, 442-51. Articles describing gene therapy techniques are Ozawa CR, Springer ML, Blau HM (2000). Annu

Rev. Pharmacol. Toxicol 40, 295-317; Furth et al. (1992),

Anal Biochem 205, 365-368; and Tang et al. (1992), Nature 356, 152-154.

BRIEF DESCRIPTION OF THE INVENTION In this invention, invertebrate transgenic animals are provided, in particular of the Drosophila melanogaster insect. These animals are characterized by expressing a CTG microsatellite, cloned from synthetic expansions, in different tissues or moments of the animal's life. Said microsatellite has been introduced stably into the genome of the recipient organism, Drosophila melanogaster, thus obtaining transgenic animals that express RNAs containing said microsatellite in a controlled manner. This expression can be controlled by using the Gal4 / UAS system. The introduced microsatellite is hereinafter referred to as "the transgene". This expression of the transgene generates a morphological and / or physiological effect on the insect that depends on the amount of expression that is induced. We call this effect phenotype. Methods for using these transgenic animals, in which a phenotype has been caused, are provided for systematic searches of compounds with a biological activity modifying said phenotype. Likewise, these transgenic animals can be used to systematically search for genes related to altered function when expressing the transgene, using genetic techniques known to a person versed in the art. Finally, these transgenic flies can be used for the validation of potential drug therapies. DETAILED DESCRIPTION OF THE INVENTION This invention provides an invertebrate animal model in which it is possible to generate a phenotype sensitized by controlled expression of a microsatellite containing the CTG sequence. This phenotype results from the expression of a transgene in a given tissue. A sensitized phenotype means that the transgenic invertebrate animal model has a phenotype whose intensity depends on the degree of expression of the transgene. This transgenic animal falls within the scope of this invention, regardless of its stage of development, provided it develops a phenotype caused by the expression of the transgene at some point in its life. Transgenic animals with the sensitized protected phenotype are characterized by having the following phenotypic characteristics: (1) the development of a phenotype whose degree of intensity depends on the degree of transgene expression (2) a change in the expression of a related gene Functionally with the transgene causes a modification in the phenotype. The transgenic animals subject to protection are invertebrate animals. Of particular interest are those of the phylum arthropods, and specifically members of the insect class. The transgenic animal subject to protection belongs to the species Drosophila melanogaster. The description made of them herein allows an expert to reproduce the invention. However, the biological material mentioned (in total, 18 Drosophila lines) is available to the public in the Drosophila Line Collection of the Department of Genetics of the Faculty of Biology of the University of Valencia, Calle Doctor Moliner, No. 50 in the city of Burjasot, province of Valencia (Spain), with Reference Numbers with the format P (60) xy for the carrier lines of a transgene with 60 repetitions and P (480) xy - Chi for the carrier lines of 480 repetitions. In both cases, x refers to the number of the animal injected while y refers to the order number of the animal that shows the phenotypic marker that contains the transgene in the offspring of the crossing to detect the transgenic flies. A critical aspect of the transgenic animals subject to protection is that the animals carry a transgene stably integrated into their genome and whose expression can be controlled in space and time, so that a phenotype is generated by the interference it causes the microsatellite expression that contains the CTG sequence. The term "transgene" describes a genetic material that has been or will be artificially inserted into the genome of the cell. Regarding the spatial expression of the transgene, this expression generally includes the imaginary discs of the larva and embryonic structures such as embryonic musculature. The transgene expresses a product that, when directed in an appropriate spatial pattern, results in a sensitized phenotype. The transgene may be a long sequence of repetitions of the CTG trinucleotide, generally more than 60, either artificially generated or present already in nature. As such, a transgene includes at least a portion of its nucleotide sequence that is substantially similar to microsatellites that contain repeats of naturally occurring CTGs, where substantially similar means a DNA sequence with a sequence identity with the natural version of these microsatellites of at least 40%, usually at least 50% and, more frequently, at least 55%, where the sequence identity is determined with the BLAST program (Basic Local Alignment Search Tool which can be found in the following internet address: http://www.ncbi.nlm.nih.gov/BLAST) in your settings by default. In embodiments of this invention, the transgene is a microsatellite that contains the CTG sequence originating from nature or having a sequence substantially similar to this one, although it may contain other intercalated sequences, have artificial or synthetic origin and carry attached sequences (coding or not ) such as a sneak gene (known in English as "reporter") or a tag (known in English as "tag"). An example of both sequences is the green GFP fluorescent protein and its variants. The transgenes subject to protection are represented by SEQ ID NO: 1 and 2 incorporated into this application. The transgene containing the CTG repeats is stably integrated into the animal's genome in such a way that its expression is controlled in space in the desired cell type. Specifically, the transgene is stably integrated into the genome of the animal behind the UAS sequences of yeast (SEQ ID NO: 3 and 4; represented as UAS-CTG). These UAS sequences serve as binding sites for the yeast Gal4 transcription factor (SEQ ID NO: 7 and 8), so that the expression pattern of the transgene is determined by the expression pattern of the Gal4 transgene. The UAS-CTG transgene may be under the control of any convenient promoter that provides the requirement of a desired expression pattern, where this promoter may be exogenous or endogenous, but will generally be endogenous. A suitable promoter is the promoter located on chromosome 2 in the Drosophila melanogaster genome that regulates GAL4 expression according to the normal pattern of the sevenless gene in the line with reference number 2023 (reference number of the University's Bloomington Drosophila Stock Center from Indiana, USA). The transgene can be integrated into the genome of this insect in a way that can be used for the activation of expression by the promoter in a direct or indirect way, that is, in such a way that the promoter exerts its activation on the transgene in cis or trans. In other words, the expression of the transgene can be mediated directly by the promoter, or by one or more transactivating factors. When the transgene is under the direct control of the promoter, that is, the promoter regulates the expression of the transgene in cis, the transgene is stably integrated into the fly genome in a place sufficiently close to the promoter and in phase with the promoter such that the cis regulation of the promoter can take place. In other embodiments of this invention where the expression of the transgene is indirectly mediated by an endogenous promoter, the promoter controls the expression of the transgene by one or more transactivating factors, usually a transactivating factor, that is, a factor whose expression is directly controlled by the promoter and which binds to a region of the transgene in an appropriate manner to activate the expression of that transgene. Any convenient transactivation factor can be employed, but the Gal4 transaction system is generally used and reference is made to this system in embodiments of this invention. In those embodiments of the invention object of protection in which the transgenic flies use the Gal4 / UAS directed expression system, the coding sequence for the Gal4 protein is stably integrated into the genome of the animal in a manner that is functionally linked. to the endogenous promoter that controls the spatio-temporal expression. An example of this type of animal is line number 2023, available at Bloomington Drosophila Stock Center at the University of Indiana, USA. (http://flybase.bio.indiana.edu/). The transgene is stably integrated in a different position in the genome, usually a random position in the genome, where the transgene is functionally linked to an upstream activator sequence (the UAS sequences), to which the transcription factor of Gal4 yeast binds and activates the expression of the transgene. Those skilled in the art commonly employ transgenic flies possessing a UAS / Gal4 transactivation system. This system is described in detail in Brand and Perrimon (1993).

METHODS TO PRODUCE TRANSGENIC FLIES PROTECTION OBJECT Protected flies can be obtained using any protocol suitable for the stable integration of the transgene into the fly genome in a manner that allows for the requirement of controlled spatial expression of the transgene, that is, , in those places where it is of experimental interest. Several strategies for the integration of the transgene with the requirement of controlled spatial expression can be employed. Generally, the methods for producing these transgenic flies involve the stable integration of the transgene into the fly genome. Stable integration can be achieved by first introducing the transgene into a cell or fly cells, for example, in the animal's embryo. The transgene is generally present in a suitable vector, such as a plasmid. The introduction of the transgene can be achieved using a suitable protocol, where suitable protocols can be: electroporation, microinjection, and the like. Following the introduction of the transgene into the cell (s), the transgene is stably integrated into the genome of the cell. Stable integration can be either site specific or random, but it is usually random. The stable integration of the transgene in the genome of The animal can be followed macroscopically by the expression of a marker gene incorporated in the chimeric DNA that includes the transgene, for example the white gene (SEQ ID: 9), in the standard manner known to persons versed in this technique.

When integration is random, the transgene is typically integrated through the use of a transposase source. Transposase is an enzyme (SEQ ID NO: 6) that catalyzes the specific integration of DNA molecules that contain the inverted terminal repeats of transposable element P, both in natural transposons and in artificial transposons. In these experimental embodiments, the transgene is introduced into the cell by a vector that includes the requirement to possess the inverted terminal repeats (of 31 base pairs) of the transposable element P of Drosophila melanogaster (SEQ ID NO: 5). When the cell in which the transgene has to be integrated does not contain an endogenous transposase source, a vector with the transposase coding sequence, for example, a plasmid called "helper", is included with the transgene plasmid that the gene for Drosophila transposase has been cloned, such as pTURBO (as described in Steller and Pirrotta, 1986). Methods for the random integration of transgenes into the genome of a Drosophila melanogaster target cell (s) are described in Spradling (1986) and in US Patent No. 4,670,388, which is included in this application as a reference. In those experimental embodiments in which the transgene is stably integrated into the fly genome, the necessary methodology for the controlled expression of the transgene during the development of the fly is also provided both in space and time. In other words, the methods for expression are provided Directed of the transgene. To obtain the desired directed expression of a randomly integrated transgene, a particular promoter can be included upstream of the transgene as a single unit in the transformation vector employed. Alternatively, a transactivator that regulates the expression of the transgene can be used. Of particular interest is the use of the Gal4 system described in Brand and Perrimon (1993). In this particular experimental embodiment, the transgenic animals subject to protection have been obtained by: (1) generation of two separate transgenic fly lines: (a) a first line expressing the Gal4 yeast transcription factor mainly in the eye, that is, under the control of an endogenous fly promoter located on chromosome 2 (such as the Drosophila line with identification number 5793 at the Bloomington Drosophila Stock Center); and (b) a second line in which the transgene is stably integrated into the cell genome and fused with a UAS domain; (2) cross the two lines; and (3) identify the progeny with the desired phenotype, that is, an observable morphological or physiological defect that can be a compound eye that has an externally rough appearance. The steps described above are of general knowledge for those people versed in this technique. See also Brand and Perrimon (1993) and Phelps and Brand (1998). The above approach is used for obtaining fertilized eggs that include a stably integrated in ^'its genome such transgene wherein the transgene is expressed in a manner spatiotemporal appropriate for the eggs result in adults exhibiting the morphological effect and / or desired physiological (phenotype). Generally, fertilized eggs are allowed to progress in their development under the conditions that lead to the desired phenotype. The phenotype of interest of animals can be modulated by varying the conditions under which animals are allowed to mature. For example, the temperature can be modified to modulate the intensity of the phenotype obtained. Embryos or larvae grown at 29-30 degrees Celsius of temperature tend to have a stronger phenotype (which can even reach the death of the animal) than those grown at 25 degrees and the phenotype of these is usually stronger than that of individuals developed at 17-19 degrees.

USES Protected flies can be used in a variety of applications, including: (1) as a tool to elucidate the altered genetic mechanism in any disease due, in whole or in part, to the expansion of sequence-based microsatellites CTG, such as myotonic dystrophies, performing systematic genetic searches of genes functionally related to the genes responsible for the phenotype caused by microsatellite expression based on the CTG sequence as described below; (2) as a tool for the systematic search for therapeutic compounds for the treatment of any disease due, in whole or in part, to the expansion of microsatellites based on the CTG sequence, such as myotonic dystrophies; and (3) as a tool for the in vivo validation of potential treatments, even obtained by any other method, of diseases that occur due to alterations in microsatellites based on the CTG sequence (that is, as model animals of human diseases such as Myotonic Dystrophy Type 1 or 2, Sca8 or HDL2). As mentioned above, the transgenic flies subject to protection are especially useful for the systematic search for compounds with a therapeutic activity against myotonic dystrophies. A potential therapeutic strategy to relieve the symptoms of patients with myotonic dystrophy is to enhance the function of human proteins homologous to muscleblind (including the MBNLl gene). That is, that the MBNLl protein not bound to CUG trinucleotides works more efficiently. Other potential therapeutic strategies consist of activating the molecular mechanisms that lead to a reduction in the size of the microsatellite expansions based on the CTG sequence object of this patent, or design synthetic peptides that compete with MBNLl in their union with the microsatellite sequences to which it is sequestered in pathological situations. In our model in Drosophila we know that when we increase the microsatellite expression based on the CTG sequence (for this we increase the level of transgene expression) the phenotypic effect is increased. Therefore, we can use experimental conditions in which we have an intermediate morphological defect and administer a battery of compounds with potential biological activity to our animal model. Those compounds that suppress the morphological defect are molecules that can potentially collaborate in the molecular function of the MBNL protein in vivo, inhibit the pathological effect of microsatellite expansions based on the CTG sequence or enhance the loss of part of the microsatellite being tested. To confirm the specificity of the observed effect, we can test the compounds with an activity in the first test in another tissue, for example in other organs such as the wing, where we can perform a quantitative test, in order to confirm the observation. We can also validate the effectiveness of a compound on muscleblind activity by testing its effect on mutant flies for the muscleblind gene. Compounds that have a positive effect on the activity of the Muscleblind protein or gene should improve the phenotype of the muscleblind mutant organism. Thus, by using the transgenic flies that are the object of protection (or cells derived from them depending on the particular type of systematic search being carried out), compounds that have an activity with respect to myotonic dystrophies can be identified or Sca8. The compounds have an activity with respect to myotonic dystrophies or Sca8 if they modulate or have any effect on at least one parameter or symptom of these diseases, such as myotonic discharges in an electromyogram or stiffness in the muscles (myotonia), where modulating activity can reduce or enhance the magnitude of the symptom, depending on the nature of the disease and the symptom. In addition, the methods for the systematic search using the flies subject to protection can be used to identify compounds that modulate the progression of the disease, for example by joining, modulating, enhancing or repressing the activity of a protein or peptide involved in the progression of the disease. With this approach we can also identify compounds that improve, relieve or even eliminate the symptoms of the disease, where such activity may or may not have to do with the pathogenicity mechanism of the disease. The systematic search for compounds, even without an effect on myotonic dystrophy or Sca8, or the testing of compounds identified by any other method, in the flies subject to protection is also of interest. Our invention allows the identification of compounds that: (1) not yet being related to modulating the activity of an element of the pathogenic pathway of the disease, have a positive effect with respect to the symptoms of the disease and as such are potentially therapeutic, by example, compounds that alleviate the myotonia of patients; (2) compounds identified by any other method (for example in vitro assays) that cause an adverse effect with respect to the disease and therefore should be avoided as therapeutic agents or (3) compounds that identified using other methods alleviate, improve or suppress totally the symptoms of the disease and are therefore therapeutic agents of choice. In the systematic search methods with the transgenic animals subject to protection, a certain amount of candidate compound is generally administered orally. After oral administration, it was determined It undermines the effect of the compound on the phenotype caused by the expression of one of the transgenes, either a microsatellite present in nature, a portion thereof, or an artificial version, provided we obtain a phenotype. The determination of the effect is generally performed by comparison with a control, that is, a transgenic fly to which the candidate compound has not been administered. The effect of the candidate compound is determined by finding out whether one or more phenotypic characteristics due to the expression of the microsatellite transgene based on the CTG sequence are enhanced or relieved in the fly under test compared to the control fly, where characteristics in the ones that can be observed a change can be the morphology of the adult's eye, its wings or legs, its behavior (ability to walk, etc.), longevity, physiology and similar characteristics. The candidate compound is orally administered to the fly generally by mixing the compound with the fly's nutrient medium, that is, water, an aqueous solution with additional nutritional elements, etc., and placing the medium in the presence of the fly, (either the larva or the adult although it is usually the adult) in such a way that the fly feeds on that medium. The administration of the compound can also use other routes of administration such as the respiratory route or injection into the hemolymph of the insect, among others. Generally, multiple mixtures with different concentrations of the compound are tested in parallel to obtain a different response with each concentration of the candidate agent. Typically, one of these concentrations serves as a negative control, for example, by not incorporating the candidate compound. The claimed methods can be adapted to high-performance systematic searches in which a large number of candidate compounds are tested in parallel using a large number of flies. By "a large number" is understands a plurality, where plurality means at least 10 or 50, usually at least 100, and more normally at least 1000, and may be between 10,000 or 50,000 or more but often will not exceed 5000. Of particular interest in some Embodiments of this invention are the use of transgenic flies that are the object of protection in high-performance systematic searches in toxicity tests, as described in US Patent No. 6,365,129 and which is incorporated by reference. In such high-performance systematic searches, the toxicity, if any, of a plurality of compounds, usually at least 10 different ones, is tested simultaneously, putting them in contact with a population of the transgenic animals subject to protection presenting a phenotype due to microsatellite expression based on the CTG sequence, and determining the effect of such compounds on animals. Such high-performance systematic searches are especially useful for finding potentially therapeutic agents for the treatment of myotonic and Sca8 dystrophies, since only those compounds that treat the disease but are not toxic enough to allow the animal to live are identified as positive for further studies. . The methods object of protection can be used in the systematic search for different potentially therapeutic candidate agents. These candidate agents cover numerous classes of chemicals, although typically they are organic molecules, preferably small organic compounds with a molecular weight of more than 50 and less than 2500 Daltons. These candidate agents have functional groups necessary for physical interaction with proteins, particularly hydrogen bonds, and typically include at least one amine, carbonyl, hydroxyl or carboxyl group, although preferably they have at least two of the functional chemical groups. Candidate agents often include cyclic or heterocyclic carbon structures and / or aromatic or polyaromatic structures substituted with one or more of the functional groups mentioned above. Candidate agents are also among the biomolecules including, but not limited to: peptides, saccharides, fatty acids, spheroids, purines, pyrimidines, their derivatives, structural analogues or combinations thereof. Candidate agents can be obtained from various sources that include collections of natural or synthetic compounds. For example, techniques are available for directed and random synthesis of a wide variety of organic compounds and biomolecules, including random collections of oligopeptides and oligonucleotides. Alternatively, collections of natural compounds in the form of extracts of bacteria, fungi, plants and animals can be obtained or produced. Additionally, libraries of natural or synthetic products, or their individual components, can be modified by conventional biochemical, physical or chemical techniques and can be used to produce combinatorial collections. Known pharmacological agents may undergo random or directed chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogues. New potentially therapeutic agents can also be created using methods such as rational drug design or structural biochemistry. Systematic searches may be directed to known pharmacologically active compounds or chemical analogs thereof, or to new agents with unknown properties such as those created by rational drug design. Candidate agents with therapeutic activity with respect to myotonic dystrophies and Sca8 can be identified on the basis of their ability to enhance one or more aspects of the phenotype of the transgenic flies subject to protection, such as a rough eye phenotype, defective wings, reduced longevity, locomotor problems and the like, as described more above. Of particular interest is the use of the methods of protection to identify therapeutic agents against myotonic dystrophies, Sca8 and HDL2 that exhibit low toxicity but are effective in enhancing the activity of the Muscleblind protein. These methods require great specificity on the part of the therapeutic agent so that, while exerting an effect on the function of the Muscleblind protein, the undesirable side effects are limited enough to allow the fly to survive. Methods of systematic searches in flies can be part of a multi-step systematic search process in which the evaluation of the efficacy (and safety) of a candidate therapeutic agent is carried out in more than one model organism. In systematic multi-step searches in which the protected transgenic flies are used, a candidate compound or collection of compounds is subjected to evaluation in a second in vivo model, for example a mouse model. Myotonic dystrophy models in mice have been generated with transgenic mice that express varying degrees of CTG expansion in the human DMPK gene and are described in detail in Mankodi et al. (2001) and in Seznec et al. (2001). In addition, a systematic in vitro search can also be used prior to the use of model animals for the disease. In these cases, the compound is first subjected to a systematic in vitro search, testing its potential as a therapeutic agent in the treatment of myotonic dystrophies. Can be used any convenient trial for a systematic in vitro search for a desirable activity to relieve or eliminate symptoms associated with myotonic dystrophies, Sca8 or other genetic diseases caused by the expansion of microsatellites based on the CTG sequence. A person versed in the subject knows the convenient essays.

IDENTIFICATION OF GENETIC TARGETS In addition to their use as model animals for the systematic search for candidate therapeutic agents, the transgenic flies subject to protection can be used to identify genes functionally involved in triggering the phenotype due to expression of the transgene being protected. Among these genes is the muscleblind gene. The genes relevant to the microsatellite expression phenotype based on the CTG sequence can be identified by performing traditional suppressor and enhancer analyzes on the flies protected. In these analyzes, the genes of the transgenic flies subject to protection are mutated to identify those that potentiate or suppress the phenotype of transgene expression. Methods for mutating genes and carrying out systematic searches of suppressors and enhancers are known to persons skilled in the art (Hays et al., 1989; Deuring et al., 1989; Fuller et al., 1989; and Rottgen et al., 1998). The genes that mutate to suppress the microsatellite expression phenotype based on the CTG sequence in a recessive manner identify potentially therapeutic proteins for the treatment of myotonic dystrophies, Sca8 and HDL2 since reducing the normal gene product of such genes would potentially alleviate disease. You can interfere with the activity of these genes through many methods that range from deleting the gene's DNA to inhibiting its transcription, translation or well its protein activity.- For systematic searches of candidate agents, small antagonistic molecules of these genes can be produced and their efficacy evaluated in the animal model by oral administration. Alternatively, large molecular antagonists can be used by gene therapy as described below. The genes that mutate to enhance the microsatellite expression phenotype based on the CTG sequence in a recessive manner identify targets to increase the activity of such genes as this would potentially lead to an improvement in the symptoms of myotonic and Sca8 dystrophies.

TEST DEVICES (KITS) Along with the transgenic flies that are the object of protection are included kits that can be used to carry out systematic search methods. Such kits include a plurality of transgenic flies of this invention, or means for producing such a plurality of flies, that is, a transgenic male fly and female fly of the present invention, the vectors carrying the required genes, such as transgenes. , a gene for the transposase gene, GAL4, etc. Flies can be confined in appropriate containers, that is, vials. The present kits may also include a nutrient medium for animals, that is, culture medium for Drosophila.

THERAPEUTIC AGENTS AND PHARMACEUTICAL FORMULATIONS The present invention also includes therapeutic agents for use in the treatment of myotonic and Sca8 dystrophies, as well as pharmaceutical formulations therefrom. The therapeutic agents of the present invention are those identified using the systematic search methods described above that show a modulating activity with respect to the microsatellite expression phenotype based on the CTG sequence (or known agents that have an effect on the expression of a gene identified as a modulator of the microsatellite expression phenotype based on the CTG sequence, where non-transgenic animals described in this application are also used for the identification of these genes) . Also included are the pharmaceutical formulations of the therapeutic agents subject to protection. In the pharmaceutical formulations or compositions of the present invention, the agents described above are formulated in pharmaceutical compositions by combination with the appropriate and accepted excipients or diluents for the preparation of drugs, which can be formulated in preparations in solid, semi-solid, liquid or gaseous form. , such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhaled and aerosols. In their pharmaceutical doses, the agents can be administered in their accepted salts for the preparation of drugs, or they can also be used alone or in the appropriate associations, as well as in combination with other active pharmacological compounds. The following methods and excipients are merely examples and are by no means limiting. For oral preparations, the agents can be used alone or in combination with the appropriate excipients or additives to make tablets, powders, granules or capsules, for example, with conventional additives such as lactose, mannitol, corn starch or potato; with additive additives, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with dispersing additives, such as corn starch, potato starch or sodium carboxymethyl cellulose; with lubricants, such as magnesium stearate or talc; and if desired, with diluents, buffering agents, moisturizing agents, preservatives or artificial flavors. The agents can be formulated in preparations for injection by dissolving, suspending them or creating emulsions in an aqueous or non-aqueous solvent, such as vegetable oils or similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. The agents can be used in an aerosol formulation to be administered by inhalation. The compounds of the present invention can be formulated in acceptable pressurized propellants such as propane, nitrogen and the like. In addition, therapeutic agents can be formulated in suppositories by mixing them with a variety of bases such as emulsifying bases or water soluble bases. The compounds of the present invention can be administered rectally using suppositories. Suppositories may include excipients such as coconut butter, polyethylene glycol and carbohydrates, which melt at body temperature but remain solid at room temperature. In the presentation of the unit dose for oral or rectal administration, such as syrups, elixirs and suspensions, each unit dose, for example a teaspoon, a tablet or a suppository, contains a predetermined amount of the composition and may contain one or more inhibitors. . Similarly, unit doses in the form of injections or intravenous administration may include the inhibitor (s) in the composition as a solution in sterile water, saline or other pharmacologically acceptable means. The term "unit dose presentation" as used herein refers to physically discrete units. appropriate as unit doses for human and animal individuals, each unit containing a predetermined amount of compounds of the present invention calculated as sufficient to produce the desired effect in association with the pharmacologically acceptable diluent, carrier or excipient. The specifications for a new presentation of the unit dose of the present invention depend on the particular compound being used and the affect to be achieved, as well as the pharmacodynamics associated with each compound in the host. Pharmacologically acceptable excipients, such as carriers, adjuvants, carriers or diluents, are available to the public. In addition, pharmacologically acceptable auxiliary substances, such as buffering agents or for pH adjustment, agents for adjusting ionic strength, stabilizers, moisturizing agents and the like are also available to the public. When the agent is a polypeptide, polynucleotide, analogue or any compound that can mimic the endogenous function of any gene identified by the use of this invention (identified using the systematic mutant search analysis protocols described above) it can be introduced into tissues or host cells by several routes, which include viral infection, microinjection, vesicle fusion or myoblast mediated gene therapy (as described in Ozawa et al., 2000). Jet injection can also be used for intramuscular administration, as described in Furth et al. (1992). The DNA is arranged by coating gold microparticles, and is distributed intradermally by an apparatus that bombards these particles against the skin, or "gene gun," as described in the scientific literature (see, for example, Tang et al. (1992 ), where gold microprojectiles are they cover with the DNA and then the skin cells are bombarded). People versed in the art will recognize that the doses may vary depending on the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. The recommended dosage for each compound can be determined by those skilled in the art using a wide variety of means. Kits with unit doses of the active agent are provided, usually in oral or injectable doses. In these kits, in addition to the containers with unit doses there will be an informative leaflet describing the use and expected beneficial effects of the use of the drug in the treatment of the disease of interest.

METHODS TO TREAT MYOTONIC DISTROPHIES, SCA8, HDL2 AND IN GENERAL GENETIC DISEASES CAUSED BY THE EXPANSION OF MICROSATÉLITES BASED ON THE CTG SEQUENCE Methods for treating myotonic dystrophies, Sca8, HDL2 and in general genetic diseases caused by expansion are also provided microsatellites based on the CTG sequence using the active agents protected. In the methods object of protection, the host that is being treated an effective amount of an active agent of the invention object of protection is administered. By "effective amount" is meant a dose sufficient to produce a desired result, where the desired result is generally an improvement or relief, including complete cessation, of one or more symptoms of, for example, myotonic dystrophy, Sca8 or HDL2 that It is being treated. The administration of the agents can be carried out in several ways, including oral, oral, parenteral, rectal, intraperitoneal, intradermal, transdermal, cutaneous, etc. You can treat a variety of guests with the methods object of protection. Generally such hosts are mammals, where this term is used in a broad sense to describe organisms within the mammalian class, which includes the group of carnivores (for example dogs and cats), rodents (for example, mice and rats) , herbivores (for example, horses and cows) and primates (for example, humans, chimpanzees and monkeys). In many embodiments the guests will be human. The following examples are offered as mere illustrations and should not be understood as limitations at all.

Examples of embodiment of the invention

A. Preparation of transgenic flies carrying the UAS- (CTG) ₅₀ and UAS- (CTG) ₄₈ or

Two synthetic expansions of the CTG trinucleotide with 60 and 480 repetitions (described respectively in Miller et al. 2000 and Philips et al. 1998), were cloned into the multiple cloning site of pUAST such that the UAS sequences of the vector were adjacent to the 5 'end of the microsatellite (Brand and Perrimon, 1993; described above). The transcription of the construct is under the control of Gal4, which has to join the UAS sequence to activate the expression of the sequence that has been cloned into the pUAST vector. The UAS- (CTG) ₆₀ and UAS- (CTG) 'so (SEQ ID Nos: 1 and 2, respectively) constructs were integrated into the Drosophila melanogaster genome by standard microinjection procedures (Spradling and Rubin, 1982). The source of transposase used to enable the integration of the UAS- (CTG) ₆ o and UAS- (CTG) 8o constructs in the genome was provided by co-injecting the pTURBO vector (as described in Steller and Pirrota, 1986).

B. Production of flies with an adult phenotype dependent on the expression of UAS- (CTG) ₆ o and UAS- (CTG) 480 transgenes

The carrier flies of the UAS- (CTG) ₆ o and UAS- (CTG) 4 _{8 constructs} or crossed independently with different Gal4-producing flies to obtain targeted transgene expression in selected tissues as described in Brand and Perrimon (1993) and his offspring were allowed to develop. Several transgenic lines were crossed, including P (480) 3.5 (reference number in the Drosophila Line Collection of the Department of Genetics of the Faculty of Biology of the University of Valencia), with the Gal4 sevenless-Gal4 production line ( sev-Gal4; reference number 2023 at the Bloomington Drosophila Stock Center of the University of Indiana. See also the Internet address http: //flybase.bio. indiana. edu). Flies were allowed to develop to adults and the observed external morphological defects were documented. Figure 1 shows the result of this experiment in which an eye composed of wild fly is compared with that of a fly expressing UAS- (CTG) or showing a phenotype of rough eyes. The images were generated by optical microscopy (AB) of adult eyes of normal individuals (Oregon R line) and eyes of individuals expressing the 48th UAS- (CTG) transgene in the eye precursors. The anterior part is on the left and the dorsal part above. The degree of phenotypic manifestation caused by the expression of the transgene, directly observable under a microscope, is variable if transgenic lines are used that express different amounts of CTG repeats, Gal4 lines that express the transcriptional activator with different intensity, as well as if different cultivation temperatures are used. Figure 2 shows a transgene expression experiment similar to that described in Figure 1 but using a different Gal4 line, in this case the GMR-Gal4 described in XX. Carrier flies of the Gal4 construct manifest a rough eye phenotype even in heterozygosis and are included in the figure as a control. This phenotype, however, is markedly increased by expressing the UAS- (CTG) transgene ₄₈ or indicating that the microsatellite expression that contains the CTG sequence of the transgene is interfering with the normal development of Drosophila's eye. Similarly, Figure 3 shows the phenotype generated when the UAS- (CTG) ₄₈ transgene is expressed or in the musculature of Drosophila melanogaster flies. For this, the line described in XX expressing Gal4 was used with the expression pattern of the myosin heavy chain gene and the offspring of the crossing were allowed to develop at 29 ° C. It is evident from the results and discussion presented above that the invention object of protection provides a valuable tool for the systematic search for potentially therapeutic agents for the treatment of, for example, myotonic dystrophies, Sca8 and HDL2. The advantages of using the transgenic flies of this invention for the systematic search of potential therapeutic agents include: the possibility of adapting the flies subject to protection protocols of high-performance systematic searches, the simplicity and low maintenance cost of said transgenic flies, the ability of flies of this invention to identify active therapeutic agents orally, rapid reproduction, and the ability of flies of this invention to produce large amounts of offspring. For all this, This invention fills an existing gap in the arsenal of tools to carry out systematic searches of therapeutic compounds because it provides a method for performing such searches in vivo and with high performance protocols. An additional significant advantage is the possibility of using the flies subject to protection to identify compounds that do not exhibit toxicity (or very low) in normal cells but are still effective in the treatment of myotonic dystrophies, Sca8 or HDL2 or other diseases in that the expansion of repetitions based on the CTG sequence is relevant. As such, the systematic search methods subject to protection provide a way to identify therapeutic agents against these diseases that exhibit low or no toxicity in normal cells. Therefore, the present invention represents an advance in the state of the art. Although the invention presented above has been described in some detail by means of illustrations and the description of examples for the sake of clarity, it is evident to people skilled in the art, and in light of such descriptions, that certain changes and modifications can be made in this invention without deviating from the spirit and scope of the following claims.

Description of the figures

Figure 1. Eye composed of a wild fly (A) compared to the rough eye caused by expression of the UAS- (CTG) ₄ 80 transgene with the expression pattern of sev-Gal4 (B).

Figure 2. Result of UAS- transgene expression

(CTG) 480 (line P (480) 1.1) in a general pattern in the precursors of the Drosophila compound eye. To control The GMR-Gal4 line expression was used and the cultures were maintained at 25 ° C. In (A) a heterozygous control individual is shown for the GMR-Gal4 construct in which it can be seen that this construct, by itself, gives a rough phenotype in the compound eye. This phenotype, however, is remarkably enhanced when GMR-Gal4 activates the UAS- (CTG) ₈ transgene or as can be seen in the image (B) by a reduction in eye size and a greater presence of collapsed omitidia.

Figure 3. Phenotype caused by transgene expression. The phenotype of the normal individuals of the OrR (A) strain is compared against the phenotype caused by expressing the UAS- (CTG) ₄ 80 transgene in the P (480) 1.1 line in the adult fly musculature, following the expression pattern of MHC-Gal4 (B). The MHC-Gal4 transgene follows the expression pattern of the muscle myosin heavy chain gene (line L82 belonging to the Drosophila Line Collection of the Department of Genetics of the University of Valencia). The offspring developed at 29 ° C.

Claims

1.- Transgene characterized in that it includes a microsatellite containing the CTG sequence, selected from SEQ ID NO: SEQ ID NO: 2, which is expressed in the Drosophila melanogaster fly according to the expression pattern of Gal4 yeast transcription factor, under direct or indirect control of a suitable, exogenous or endogenous promoter, said expression causing a morphological or physiological effect on the phenotype of the measurable fly proportionally to the level of transgene expression.

2. Transgene according to claim 1 characterized in that it is expressed under the control of any endogenous promoter capable of regulating the expression of Gal4 according to the normal Drosophila melanogaster pattern selected among others, from genes such as the sevenless gene or the apterous gene.

3. Transgene according to any of claims 1 or 2 characterized in that it is expressed through one or more transactivating factors, preferably belonging to the Gal4 transactivation system.

4.- Method of obtaining transgenic Drosophila melanogaster flies characterized by: a) Generating a first line of transgenic flies that express the Gal4 yeast transcription factor, under the control of any endogenous Drosophila melanogaster promoter, preferably in the eye of said fly. b) Generate a second line of flies in which the transgene of claims 1 to 3 is stably integrated and fused with the UAS sequence c) Cross both fly lines d) Select the progeny with the desired phenotype

5. Method according to claim 4 characterized in that the stable integration of the transgene, specific or random, in at least one cell of the fly, preferably in an embryo state, is done through a suitable vector, preferably a plasmid, according to any of the technologies known in the sector, among others electroporation and microinjection.

6. Method according to claim 5 characterized in that when the integration of the transgene is random and the cell whose genome is integrated has an endogenous source of transposase, the vector containing the transgene also has the terminal inverted repeats of 31 base pairs of the transposable element P of Drosophila melanogaster.

7. Method according to claim 5 characterized in that when the integration of the transgene is random and the cell whose genome is integrated does not possess an endogenous source of transposase, the vector containing the transgene is injected together with a transposase coding sequence. from Drosophila.

8. Method according to claim 7, characterized in that the vector containing the transgene is preferably plasmid pTURBO.

9. Drosophila melanogaster transgenic flies obtainable according to the method of any of claims 4 to 8 characterized by having a genome containing the transgene of claims 1 to 3 which is expressed in different tissues and at different times of development of flies using the Gal4 / UAS expression system, resulting in a recognizable phenotype.

10.- An animal model for the identification, individually or simultaneously, of candidate compounds useful for the treatment of any disease in whose pathological state the presence of microsatellite expansions based on the CTG sequence is relevant, characterized in that it comprises: a) Administering said candidate compound to the Drosophila melanogaster transgenic flies of claim 9. b) Measuring the effect of said candidate compound on the phenotype generated in transgenic flies expressing the transgenes of claims 1 to 3 in a given tissue. c) Compare the phenotype of the transgenic flies that express said transgenes in a given tissue with the phenotype of transgenic flies that express the same transgenes under identical conditions but did not receive the candidate product. d) Identify, as potentially therapeutic compounds, those that are capable of causing an increase or decrease in the phenotype, which is indicative of a potentiating or reducing activity, respectively, of the pathogenic capacity of the transgene.

11.- A model for the test and / or validation of candidate compounds useful for the treatment of any disease in whose pathological state the presence of microsatellite expansions based on the CTG sequence, identified in vivo or in vitro, by any system is relevant. biochemical or biological, including the model of claim 10, comprising: a) Administering said candidate compound to the Drosophila melanogaster transgenic flies of claim 9. b) Measuring the effect of said candidate compound on the phenotype generated in transgenic flies expressing the transgene of claims 1 to 3 in a given tissue. c) Quantitatively compare the increase, reduction or alteration of the phenotype of the transgenic flies that express the transgene of claims 1 to 3 in a given tissue with the phenotype of transgenic flies that express said transgene under identical conditions but did not receive the candidate product. d) Identify, as potentially therapeutic compounds, those that are capable of causing an increase or decrease in the phenotype, which is indicative of a potentiating or reducing activity, respectively, of the in vivo pathogenic capacity of the transgene of claims 1 to 3. e) Qualitatively compare the phenotype of the transgenic flies expressing the transgene of claims 1 to 3 in a given tissue with the phenotype of transgenic flies expressing said transgene under identical conditions but did not receive the candidate product so that the appearance of a phenotype unrelated to the phenotype caused by expression of the transgene of claims 1 to 3 is indicative of a side effect of the compound tested.

12. A model according to any of claims 10 or 11, characterized in that the candidate compound is administered to Drosophila melanogaster transgenic flies in a nutritious medium.

13. A model according to any of claims 10 or 11, characterized in that the candidate compound is administered to the Drosophila melanogaster transgenic flies by respiratory route.

14. A model according to any of claims 10 or 11, characterized in that the candidate compound is administered to the Drosophila melanogaster transgenic flies by injection into the body cavity.

15. A model according to any of claims 10 to 14, characterized in that the disease is selected from myotonic dystrophy type 1 and 2, spinocerebellar ataxia 8 or HDL2 disease.

16. An animal model for identifying genes that potentially modulate the activity of the transgenes described in claims 1 to 3 which comprises: a) Producing or introducing, by means of genetic crosses, a mutation in the genome of the transgenic fly of the claim 9. b) Determine if said mutation has an effect of increase, reduction or alteration on the phenotype generated by the expression of the transgene of claims 1 to 3. c) Identify the mutated gene that was introduced in step (a) or in which the mutation occurred.