MX2011009743A - Compositions and methods for stimulating the neural differenttiation via dystrophin dp71?78-79. - Google Patents

Abstract

The present invention is referred to nucleotide molecules, methods and compositions for stimulating the neural differentiation. The invention shows the implication of the dystrophin DP71?78-79 protein in the neural differentiation process, providing a novel and useful method for stimulating the neural differentiation in cell lines or in subjects, particularly cell lines such as PC12 and in human subjects with certain degree of mental deficiency produced by diseases where a neurodegeneration is present such as Duchenne Muscular Dystrophy (DMD). So that, the DP71?78-79 protein of the present invention has been cloned in a molecular vector, resulting in a vector best know as pcDNA4/HisMax-TOPO-TA, the mutation lacking the genes 78 and 79 of the Dystrophin Dp71, but maintaining the exons 1 and 6 to 77.

Description

Compositions and methods to stimulate neuronal differentiation via dystrophin DP7 A78-79 Field of the invention.

The present invention relates to nucleotide molecules, methods and compositions that stimulate neuronal differentiation. The invention shows the involvement of the protein Dystrophin? 71? 78 79 in the process of neuronal differentiation, providing the basis for the development of a new therapeutic method. More specifically, the present invention relates to the use of Dop71A78.79 Dystrophin to stimulate neuronal differentiation in cell lines, stem cells in general and neural stem cells in particular, in animal models or in individuals, particularly cell lines such as PC12 and in humans that present damage in the functioning of the nervous system and / or a certain degree of mental retardation caused by trauma or diseases where there is a neuronal degeneration such as Muscular Dystrophy of Duchenne (DMD).

BACKGROUND OF THE INVENTION Neural degeneration is an important consequence of a wide range of conditions that occur in humans causing different degrees of mental retardation, such is the case of the disease known as DMD.

Duchenne muscular dystrophy (DMD) belongs to a group of inherited diseases that are characterized by progressive muscular degeneration with some degree of mental retardation and cardiomyopathy1. This dystrophy is a recessive disorder linked to the X chromosome, caused by mutations in the DMD gene, which codes for a dystrophin family2. DMD affects approximately one in every 3,500 children. In patients with DMD, the primary biochemical defect is the absence of full-length dystrophin (427 kDa), whereas Becker's muscular dystrophy (BMD), a less severe condition, is caused by mutations that lead to reduced expression of dystrophin or truncated isoforms, with a partial function of the protein. The important abnormalities that occur in muscle cells deficient in dystrophin occur at three different levels: 1) alterations in the permeability of the cell membrane; 2) the deregulation of calcium homeostasis, a process that is critical for many aspects of cellular function; and 3) the increase in susceptibility to oxidative toxins3.

DMD is characterized by a delay in the development of motor parts and muscular weakness, with the loss of muscle coordination evident between 18-20 months of age. At a later stage, patients are confined to a wheelchair and die around the age of twenty due to respiratory complications, muscle weakness, cardiac dysfunction with cardiomyopathy and / or cardiac conduction abnormalities3. In addition to myopathy, in patients with DMD, there are other non-muscular manifestations such as heart disease4, mental retardation5,6, cognitive deficit7, abnormalities in the retina8,9,10 and possible ear defects11. Other characteristics observed in children born with DMD is the high level of the muscle isoform of creatine kinase in the serum, an enzyme that is released in the muscle that has suffered damage, as well as the necrosis or degeneration and regeneration of the muscle fibers; eventually, the regenerative capacity of the muscles is lost and the fibers are gradually replaced by fibrous and adipose connective tissue giving an appearance of pseudohypertrophy followed by muscular atrophy3. It has also been reported that patients with skeletal muscle disease can develop a serious disease of the heart4,12,13 and vice versa, where mutations in the dystrophin gene sometimes result in cardiomyopathy without evidence of skeletal myopathy14,15, 16 The DMD gene.

The DMD gene was identified in 198717 and corresponds to the largest gene described to date, since it has a size of 2.5 Mb and consists of 79 exons18,19,20. This gene is located on the short arm of chromosome X at the Xp21 locus and codes for a dystrophin family (figure 1) 2. Mutations in the dystrophin gene correspond to eliminations that result in the lack or mutated forms of this protein2, with the majority of the deletions detected in the DMD gene occurring in two mutation sites or regions called "hot-spots" 21, 22 The nature of these mutation sites is unknown, although it is believed that the chromatin structure at the Xp21 locus may influence the generation of these mutations. The first "hot spot" mutation site covers exons 45 through 5323 with several breakpoints in introns 44, 45, 47, and 5024.25, removing part of the domain from spectrin-like repeats, while the second site comprises exons 2 to 20 with many breakpoints in introns 1 and 7, affecting part or all of the actin binding domain and a portion of the spectrin-like repeats26,27. A third of the cases of DMD are caused by small eliminations or point mutations that introduce a premature stop codon28,29. These small deletions and mutations appear to be distributed throughout the gene 29 · 6 · 30; Although these mutations can produce a normal amount of truncated proteins, usually the protein is not detected or only a small amount is detected, suggesting that the corresponding transcripts or the truncated proteins are unstable17.

The size of the deletions within the DMD gene does not correlate with the observed clinical phenotype. This observation can be explained by the Monaco3 reading frame theory, which argues that if a deletion results in the expression of a truncated protein without changing the reading frame, a small isoform of dystrophin is produced that may be functional , scenario compatible with the BMD. On the other hand, if the mutation creates a change in the reading frame and a premature termination of the translation occurs, it results in the synthesis of a modified protein that is additionally a truncated protein. This generally leads to the absence of dystrophin or the expression of a low level of the protein and therefore a DMD phenotype. With the use of this reading frame theory and knowledge of the DMD gene, in many cases, it has been possible to predict whether a child will develop DMD or BMD21, however, it is convenient to consider that there are exceptions to the reading frame rule3.

The dystrophin.

Dp427 dystrophin is composed of four main domains: an NH2-terminal domain that interacts with the actin filaments of the cytoskeleton, a central domain of 24 repeating triple-helix spectrin-like, a domain rich in cysteine and a COOH-terminal domain ( Figure 1) 31 · 32 · 3. The repeats of alpha helix similar to spectrin are interrupted by four hinge regions rich in proline that participate in the flexibility of dystrophin22. The repeating region similar to spectrin is followed by a WW domain that modulates the binding of signaling and regulatory proteins; this region mediates the interaction between β-dystroglycan and dystrophin33,34. The domain rich in cysteine has two "motifs" EF that are similar to those of a-actinin and that can bind intracellular calcium35, as well as a ZZ domain, similar to zinc fingers, which contains a number of conserved cysteine residues that are binding sites for divalent cationic metals such as Zinc36. The ZZ domain of dystrophin binds to calmodulin in a calcium-dependent manner37 and is not required for the binding of β-dystroglycan38. Within the COOH-terminal domain of dystrophin, two "coiled co / 'f domains are found that are binding sites for distrobrevins, sintrophins and other proteins associated with dystrophin39'40.

Promoters of the DMD gene.

The mRNA of the dystrophin family is transcribed from seven promoters located in different regions of the gene, resulting in tissue-specific expression of several isoforms of dystrophin (Figure 1) 3. The total length of the mRNA of the DMD gene is 14 kb, which is expressed predominantly in skeletal and cardiac muscle, with small amounts found in the brain2. The expression of Dp427 is the result of transcripts initiated from three different promoters in the 5 'region of the gene, which are regulated independently. The B promoter regulates the expression of Dp427 in cortical and hippocampal neurons of the brain, the P promoter in Purkinje cells and in skeletal muscle, and the M promoter in skeletal and cardiac muscle and presents low levels of expression in some glial cells41, 3. Nishio et al.42 described the promoter (L) upstream of the muscle promoter, as active in lymphoblastoid cells; however, some authors have questioned its functionality as an authentic promoter of the DMD43 gene. There are internal promoters, which are located within introns and regulate the expression of four small isoforms of dystrophin. These promoters are Dp260-R that is expressed in retina, Dp140-B / K that is expressed in brain and kidney, Dp1 16-S in Schwann cells and Dp71-B / U with ubiquitous expression and whose levels are elevated in brain3, 41, each of these promoters are located in introns 29, 43, 55 and 62, respectively, to generate the 260 kDa (Dp260) 44, Dp14045, Dp1 1646 and Dp7147 48 proteins. The Dp71-B / U promoter it also codes for a 40 kDa protein, Dp40, this isoform presenting an alternative processing in which exons 71 to 7949 are eliminated. Small transcripts, with the exception of Dp40, are encoded by the same exons of the 3 'region of the DMD gene and the product of the translated proteins contain the binding sites required for DAPs (dystrophin-associated proteins) complex proteins 50,3. The common characteristic of all isoforms of dystrophin is the conservation of the motif WW, the EF region and the coiled-coif regions present in the COOH-terminal (figure 1), with the exception of the Dp71 and the Dp40 that present half of this "motif." The cellular and molecular function of these isoforms has not been elucidated yet, but they could be involved in the stabilization and function of the DAPs3 complex.

Post-transcriptional modifications of dystrophin mRNA.

The mRNA of the dystrophin family has alternative processing in the 3 'region and generates a large number of isoforms with differences in the COOH-terminal region51,52. The Dp140 isoform in human brain and kidney presents an alternative processing in exons 71, 78 and 71 to 74 generating at least four isoforms53. In brain and other tissues, Dp71 also has alternative processing in the same exons, so that multiple products of Dp71 of 70-78 kDa54.55 and a protein of approximately 58 kDa designated Dp71An0 55 are generated. The alternative processing of Dp71 in exon 71 does not change the open reading frame while the loss of exon 78 changes the open reading frame resulting in the replacement of 13 hydrophilic amino acids from the COOH-terminus by 31 new amino acids with hydrophobic properties, called the "founder" sequence ( 2) 56. This isoform of Dp71 that lacks exon 78 is called Dp71f or Dp71 b and the isoform that has exon 78 is called Dp71 d or Dp71 a. It has been found that the alternative processing of exons 71 and 78 of Dp71 regulates its nuclear or cytoplasmic localization57,58,59; whereas the Dp71f isoform increases its expression levels and migrates from the cytoplasm to the varicosities and the growth cone during the differentiation process of the PC12 cells, the levels of the Dp71 d isoform remain constant throughout the cell and are concentrated in The nucleus during this process, suggesting that these proteins play an important role during the differentiation of PC1259 cells.

Post-translational modifications of dystrophin.

The COOH-terminal domain of dystrophin has several potential phosphorylation sites and these sites are phosphorylated both in vivo and in vitro by several kinases, including MAPK (p44erk1 and / or p42mpk), protein kinase p34cdc2, cAMP-dependent kinases (PKA) and cGMP, casein kinase II (CK-II), GSK-3, CaM KM and probably by other membrane-associated kinases, and is dephosphorylated by the calcineurin phosphatase type 2B60. Phosphorylation of dystrophin by PKA increases the interaction with actin, whereas phosphorylation by CK-II and protein kinase c (PKC) inhibits this interaction61; likewise, the phosphorylation of dystrophin by CaM KM inhibits its interaction with sintrophin62. Another mechanism for regulating the interaction of dystrophin with the proteins of the DAPs complex is the post-translational modification of the components of the DAPs complex, for example, the phosphorylation of the COOH-terminal region of β-dystroglycan prevents its interaction with dystrophin63 Calderilla-Barbosa et al.64 suggest that the phosphorylation of threonine 3685 present in exon 79 determines the nuclear localization of Dp71a; therefore, the function of the dystrophins and the components of the DAPs complex can be regulated by multiple mechanisms.

Complex of proteins associated with dystrophin.

In skeletal muscle the NH2-terminal domain of Dp427 binds to cytoplasmic actin while the cysteine-rich region and the COOH-terminal domain are important for the interaction of this dystrophin with a complex of transmembrane glycoproteins and cytoplasmic factors known as protein complex associated with dystrophin or DAPs (figure 3) 1, 3. This complex consists of three distinct sub-complexes: the distroglycan complex, the sarcoglycan-sarcospan complex and the cytoplasmic complex that contains dystrophin65. The components of the protein complex associated with dystrophin include dystrophin, a, ß, d, e, yy? -sarcoglycan, ay ß-dystroglycan, a and ß-distrobrevin, a and ß-sintrophin, sarcospan, caveolin-3, nitric oxide neuronal synthase (nNOS) and calmodulin3,66,1. The expression and distribution of these proteins varies depending on the type of tissue66. Sintrophins and distrobrevins recruit signaling elements such as nNOS, Grb2 and also participate in ion channel clustering39,67; β-dystroglycan also binds to the adapter protein Grb268,69,70 and caveolin-371. Grb2 binds to p125FAK (focal kinase adhesion) suggesting that the components of the DAPs complex have signaling and platform functions69.

The skeleton of the DAPs complex consists of a and β-dystroglycan, where the β-dystroglycan has a single transmembrane domain and is inserted inside the plasma membrane with the COOH-terminal towards the cytoplasmic side3, this region is rich in proline and medium the interaction with dystrophin through the domain WW 33,72,34. This union is stabilized by the "motifs" EF present in the cistern-rich region'38,72. The NH2-terminal region of β-dystroglycan interacts with the COOH-terminus of α-distroglycan, a component of the extracellular matrix, which in turn interacts with several components of the basement membrane such as laminin, heparan sulfate, agrin, neurexin and biglycan . Neurexin mediates the interaction of the neuronal post-synaptic membrane, through a-dystroglycan, with the pre-synaptic membrane73.

Mutations of dystrophin in the cysteine-rich and COOH-terminal domains affect their interaction with β-dystroglycan and other members of the DAPs complex, resulting in a severe dystrophy phenotype (DMD); in contrast, mutations in the NH2-terminal region of actin binding cause a less severe phenotype (BMD) 1. This is probably due to the presence of other actin-binding sites, one of which is within the repeating spectrin-like region, from 1 1 to 17 that binds to -actin74 and another in the "coiled" region. -coif that interacts with F-actin.75,76 The eliminations in the spectrin-like repeats, without change in the reading frame, do not have serious consequences.77 This observation suggests that the spectrin repeats act as a spacer region between the NH2 domain. -terminal of binding to actin and the domains rich in cysteine and COOH-terminal dystrophin, the elimination of this region does not affect the function of the protein3.

Mutations in the genes of some members of the DAPs complex also cause different types of muscular dystrophy1; for example, the absence of one or several sarcoglycans causes different phenotypes of dystrophy66. It has also been observed that the levels of the components of the DAPs complex are decreased in the muscle of patients with DMD, in mdx mice that have lost the full-size dystrophin and in mdx-3cv mice that do not express any of the dystrophin 78. The alteration of the proteins of the DAPs complex that lead to different forms of dystrophy suggests that the connection of the cytoskeleton with the extracellular matrix is important to maintain the viability and function of the muscle cells. The exact function of dystrophin is not yet clear; however, according to previous studies, it is postulated that dystrophin plays an important role in the connection between the cytoskeleton and the extracellular matrix79,80, maintains cellular stability, participates in signal transduction and in the protection of muscle fibers. of the damage induced during contraction3,66.

Dp71 dystrophin.

Dp71 dystrophin corresponds to the main product of the dystrophin gene that is expressed in tissues of the human brain54,81, 55 and of the rat brain 48,58 although it is also abundant in a great variety of non-muscular tissues 47,48,56. This dystrophin has a specific NH2-terminal region of seven amino acids that is fused with the domain rich in cysteine and COOH-terminal, a region crucial for the binding of the proteins of the DAPs82 complex. The seven amino acids of the NH2-terminal region are encoded by exon 1 of Dp71 and the remaining amino acids are encoded by the exons from 63 to 79 with possible alternative processing of exons 71, 78 or 71 to 74 54.55. Although Dp71 does not have the NH2-terminal domain of actin binding, its unique NH2-terminal region has a "motif that is able to interact with the actin cytoskeleton and the alternative processing of exons 71 and 78 does not alter this interaction 83,58,84 A working group showed that the subcellular localization of Dp71f in Müller glial cells is not random in the cytoplasm, but is found in groups that coincide with high concentrations of actin filaments85. This same distribution of Dp71f was previously described by Howard and col.86 in mouse and human retina, where they found that Dp71f is located very close to the plasma membrane. Likewise, it has been shown that Dp71 is associated with the plasma membrane in HepG2, HeLa87 and astrocyte cells88, as well as with actin filaments in the cell line C2C1289.

Expression and function of Dp71 dystrophin.

The exact function of Dp71 is not yet known, however, several studies have demonstrated the importance of Dp71 in different cellular processes. For example, the absence of Dp71 alters the expression and localization of the DAPs complex in the brain90,78,91, kidney92 and retina93. In this same context, it has been described that Dp71 is important for the organization and / or stabilization of the components of the DAPs complex, water and potassium channels, and to maintain the morphology of the plasma membrane94,95,96,93. Recently, it has been reported that the absence of Dp71 contributes to the severity of mental retardation in patients with DMD and BMD97,98. The association of Dp71 with the proteins of the DAPs complex such as ß-dystroglycan72.85, 93.99.100_ distrobrevin101'102 and sintrophin50,85,102; its interaction with actin84,85,93,95 and with the plasma membrane87,86, demonstrate the importance of the Dp71 isoforms in the organization of the components of the DAPs complex. Another interesting fact is that the transgenic expression of Dp71 in mdx mice, which naturally do not express Dp427 dystrophin, restores the components of the DAPs complex in the membrane; however, the phenotype of muscular dystrophy is not corrected103,104.

The expression of Dp71 seems to be associated with morphogenetic events and with terminal differentiation105. The Dp71 mRNA shows a slight reduction during the differentiation of human fetal muscle cells (myoblasts) and an increase in the amount of mRNA of Dp427106. Although it is a ubiquitous isoform, Dp71 is not expressed in adult skeletal muscle47,56; its expression in muscle tissue is only observed in myoblasts82,107 reducing the level of expression during the process of differentiation of muscle cells108. In contrast, Dp427 is not expressed until the cells initiate myogenic differentiation and it is the isoform that is expressed mainly in mature muscle fibers109,110, indicating that the expression of these isoforms in muscle cells is very well regulated89. The negative regulation of Dp71 expression during muscle cell differentiation occurs, at least in part, at the transcriptional level as a result of the differential expression of the transcription factors Sp1 and Sp3108; however, it has also been seen that the Dp427 and Dp71 proteins are present at low levels in undifferentiated muscle cells and both proteins increase their expression during the differentiation process106. On the other hand, the expression of Dp71 in brain increases during fetal development and the maximum level of expression is observed in adult brain58. The importance of the Dp71 isoforms during the process of neuronal differentiation of cells PC1259, 1111 and N1 E-1 15112 has also been described.

Isoforms of Dp71 dystrophin.

At present, six isoforms of Dp71 have been described in different species, derived from alternative processing. Dp71 (NM_004015), is the complete isoform that presents all the exons of Dp71; Dp71 a (NM_004017), this isoform of Dp71 has an alternative processing in exon 71, where the elimination of exon 71 does not change the reading frame so that the COOH-terminal is equal to Dp71 (figure 2); Dp71 b (NM_004016), previously Dp71f, where this isoform lacks exon 78. The processing of exon 78 changes the reading frame and the result is the replacement of the 13 hydrophilic amino acids of the COOH-terminal of normal Dp71 by 31 new amino acids with hydrophobic properties56; Dp71 ab (NM_004018), isoform that has the alternative processing of both exon 71 as well as exon 78, where the carboxyl terminus is equal to that of Dp71 b (Figure 2), and Dp71 c (NM_001005246) lacking exons 71 -74. An isoform of Dp71 has also been identified without exons 71 to 74 and 78, called Dp7lA1 1055- In addition, the rat Dp40 sequence has been reported49, which is expressed from the Dp71 promoter and comprises exons 63-70 , as well as the human Dp40 sequence (NM_004019).

PC12 cells: a study model for Dp71 dystrophin.

Our working group has used PC12 cells as a cellular model for the study of Dp71. PC12 cells come from a pheochromocytoma of the rat adrenal gland and have the ability to stop their proliferation and spread neurites in contact with neural growth factor (NGF). These cells are small, grow grouped in suspension, synthesize catecholamines (dopamine, norepinephrine) and in response to NGF develop a phenotype similar to the sympathetic neurons of the peripheral nervous system, this phenomenon being reversible113,114. To date it is known that PC12 cells express different isoforms of Dp71 both in differentiated cells and in undifferentiated cells, an isoform without exon 71 designated Dp71d (Dp71a) (NM_001005244); Dp71f (Dp71 ab) (NM_012698) lacking exons 71 and 78 59; the Dp71 c isoform (NM_001005246), which does not show exons 71 to 74; Dp40 that does not present exons 71 to 79115.1 6; and in our laboratory we find the expression of a new isoform of Dp71, which we call Dp71e, which has a different COOH-terminal117. This new isoform of Dp71 presents an alternative processing in which 34 base pairs are incorporated, between exons 77 and 78, with a stop codon in triplet 1 1 that prevents the translation of exons 78 and 79; this region codes for 10 amino acids with characteristics totally different from the dystrophin groups "d" and "f (Dp71 a and Dp71 ab).

PC12 cells express, at the protein level, the Dp71 d (Dp71a) and Dp71f isoforms (Dp71ab) 59. It has been observed that there is an increase in both the mRNA expression and the Dp71 protein during the differentiation of PC12118'59 cells. These isoforms have a differential expression and localization; whereas Dp71ab increases its expression and is located in the cytoplasm, varicosities and growth cones during the differentiation process, where the expression of Dp71a remains constant and is located both in the nucleus and in the cytoplasm of undifferentiated cells, concentrating in the nucleus during differentiation induced by NGF59.

It has also been reported that the neuronal differentiation of PC12 cells is affected by the inhibition of the expression of the Dp71111 isoforms, probably due to a reduction of the integrin / Dp71ab adhesion complexes described by Cerna84, suggesting that the expression Normal isoforms of Dp71 is important for adhesion and differentiation of PC12 cells. On the other hand, it has been reported that Dp71a localized in the nucleus is associated with nuclear matrix proteins, an association that is modulated during the differentiation of PC12119 cells; likewise, Dp71 forms a complex with DAPs in the nucleus of PC12 cells and the inhibition of Dp71 expression alters the expression and localization of these proteins100.

Recently, we report that the Dp71ab isoform forms a complex with β-dystroglycan, al-sintrophin, β-distrobrevin and α-, β- and β-sarcoglycan in undifferentiated PC12 cells, whereas in differentiated cells the composition of the complex changes, in where Dp71 ab is associated only with β-dystroglycan, al-sintrophin, β-distrobrevin and d-sarcoglycan; In addition to these proteins, the nNOS protein is also recruited to the complex, suggesting that Dp71ab may be involved in signal transduction during the differentiation of PC12 cells with NGF102. Likewise, our group described the role of Dp71 in the myoneural synapse using a co-culture model between PC12 cells and myotubes of L6 cells. In this model, Dp71a is located in the nucleus of PC12 cells and structures similar to the Golgi complex; in contrast, Dp71ab is concentrated in the neurite and cytoplasm terminals120. All these evidences suggest that some of the Dp71 isoforms are involved in processes related to neuronal differentiation.

Although DMD is an important hereditary disease, there are relatively few works related to the therapy of this disease being the main ones related to gene therapy. This is largely due to the ignorance that prevails in our days of the mechanisms of regulation of the expression of the DMD gene, of the function of the different dystrophin and of the domains that make up these proteins. . In this field the patents US5239060, US2008 / 0044393, EP1366160 and US2010 / 0105054 describe therapeutic and / or diagnostic methods for DMD employing the dystrophin gene, while patent US201 / 0183890 refers to the use of inhibitory molecules of Dp71 Dystrophin to modulate angiogenesis in diseases related to abnormal neovascularization; However, to date, there continues to be a need for effective therapies that allow the restoration of nervous system function damaged by said disease.

Differentiation of PC12 cells.

The PC12 cell line is the best characterized cellular model for the study of neuronal differentiation. NGF induces arrest of the cell cycle, prevents apoptosis in cells deprived of serum and stimulates neuronal differentiation of PC12 cells, resulting in the extension and growth of neurites. NGF binds to its TrkA receptor by activating a series of kinases including Erk1 / 2 and Akt, to stimulate differentiation and promote the survival of neuronal cells, respectively113,114,121'122. Neurotrophins play an important role in the regulation of neuronal survival, cell growth, differentiation and neuronal plasticity. In particular, NGF is widely used for the differentiation of pci cells 259,100,102 '113 · 114.

The arrest of the cell cycle occurs by the production of nitric oxide (NO) in response to NGF123; this is suggested because it has been shown that the inhibition of NO production prevents the arrest of the cell cycle and therefore, the differentiation of PC12 cells is inhibited; this NO signal seems to act through p53 and p21WAF1124. Another of the important factors involved in the differentiation of PC12 cells induced by NGF is the activation of the Erk1 / 2125,126 pathway. The mechanism of activation of the RTKs (TrkA) begins with the autophosphorylation of the receptor when interacting with the NGF and the recruitment of adapter proteins such as Shc and Grb2 to the receptor that allows the activation of Ras by means of GTPases; Activated ras recruits Raf kinase to the membrane, which in turn stimulates the activation of the Erk / MAPK pathway, which is crucial for the differentiation of PC12 cells, since blocking the activation of Erk / MAPK It inhibits the induction of neurites and the constitutive activation of the Erk / MAPK pathway allows the differentiation of PC12 cells in the absence of NGF 22. Activated Erk translocates to the nucleus and phosphorylates various transcription factors such as the TCF factor (ternary complex factor ), Phosphorylated TCF and its transcriptional co-activator SRF (serum-response factor) stimulates the transcription of a series of immediate response genes such as c-fos (figure 4) 127, Egr 28, among other factors, thus allowing neural differentiation of PC12 cells.

Therefore, it is important to provide methods and therapeutic compositions comprising active principles that stimulate neuronal differentiation as a strategy for the treatment of conditions where there is damage to the functioning of the nervous system or mental retardation, as for example in DMD.

Objectives of the invention.

Therefore, one of the objectives of the present invention is to provide protein sequences of the protein? 71? 78 7g, in order to stimulate and / or generate neuronal differentiation.

Another objective of the invention is to provide coding sequences for the protein? 71? 78.79, which allow cloning and / or expression in various vehicles for the purpose of producing said protein.

Another objective of the invention is to provide compositions comprising the protein Dp71A78 79, for the treatment of conditions where there is damage to the nervous system, as for example in DMD.

Another objective of the invention is to provide methods for the treatment of conditions where damage to the nervous system occurs, such as in DMD, based on the stimulation and / or generation of neuronal differentiation.

Brief description of the figures.

Figure 1. Schematic representation of the DMD gene and the dystrophin family. The Gen DMD covers 2.5 Mb and has 7 promoters that code for a family of proteins called dystrophins and named according to their molecular weight.

The full-length dystrophin is transcribed from 3 independent promoters and the mRNA has a length of approximately 14 kb. He mRNA codes for a 427 kDa protein with differences in the NH2-terminal region. The three products are named as Dp427 (B), Dp427 (M) and Dp427 (P) to refer to their tissue-specific expression pattern; B: hippocampus and cortex, M: skeletal and cardiac muscle, and P: Purkinje cells. Small isoforms are expressed from internal promoters that code for the tissue-specific isoforms Dp260 (retina), Dp140 (brain and kidney), Dp1 16 (peripheral nervous system) and Dp71 / Dp40 (ubiquitous expression). The 4 main domains of Dp427 are indicated as AB: actin binding domain, RD: 24 repeats similar to spectrin, CR: region rich in cysteine, and CT: COOH-terminal. Taken from Blake et al. | Errorl Marker not defined. with a few modifications.

Figure 2. Schematic representation of the exons of Dp71 a, Dp71 ab, Dp71e and Dp71A78.7g. A) Comparison of Dp71A78_79 with the Dp71a, Dp71ab and Dp71e isoforms. Dp71A78 79 presents exons 1 and 63-77 of Dp71 and lacks exons 78 and 79. Dp71A7879 does not affect any of the dp71 domains involved in the interaction with DAPs129,130. The interaction sites with ß-dystroglycan, sintrophins and distrobrevins are indicated as ß-Dg, Syn and Db, respectively. B) Amino acid sequence of the COOH-terminal region of Dp71 a, Dp71ab, Dp71 e and Dp71A78 79 corresponding to the final part of exon 77 and of the specific exons of each isoform. 1: Unique Exon of Dp71; H: Histidine tag and X: Xpress epitope fused to the vector pcDNA4 / HisMaxTOPO-Dp71A78 7g; I77: sequence of intron 77 present in Dp71 e. WW, EF1 / EF2, ZZ and CC1 / CC2 (Coiled-coil): domains of dystrophin present in all products of the DMD gene.

Figure 3. Complex of proteins associated with dystrophin. The NH2-terminal region of dystrophin binds with the actin cytoskeleton and the COOH-terminal region with a group of cytoplasmic, membrane and transmembrane proteins known collectively as dystrophin-associated protein complex (DAPs). This protein complex is classified into three sub-complexes: i) cytoplasmic: it includes the sintrophins (syn), distrobrevins (DB), nNOS, calmodulin and Grb2, ii) sarcoglycan-sarcospan: it is formed with sarcoglycans (a, ß,?, d) and sarcospan (ss) and iii) distroglycan: formed by a-dystroglycan that binds to extracellular matrix proteins and β-dystroglycan which in turn interacts with dystrophin, allowing a connection between the actin cytoskeleton and the extracellular matrix. The DAPs complex varies depending on the cell type or tissue and the isoform of dystrophin. Taken from Blake et al.39 with some modifications.

Figure 4. Cascade signaling induced by NGF. NGF binds to its TrkA receptor and activates a series of proteins until the phosphorylation of Erk1 / 2. Activated Erk1 / 2 is translocated to the nucleus and activated by indirect phosphorylation to various transcription factors such as TCF and CREB that are essential for neuronal differentiation of PC12 cells. On the one hand, NGF induces the temporary activation of the Ras and Erk pathway (B-Raf-MEK-Erk) that is not sufficient for neuronal differentiation of PC12 cells and, on the other hand, activates the M-Ras-Erk pathway. constant way, which is necessary for the process of neuronal differentiation. The activation of Rap-1 participates in cell adhesion, required for differentiation, through integrins. Cerna et al84 reported that Dp71 ab forms part of the adhesion complex with integrins in PC12 cells. Taken from Sun et al.122 with some modifications.

Figure 5. Map of expression vectors pcDNA4 / HiMax-TOPO-TA and pcDNA3.1 / V5-His-TOPO-TA. A) Vector pcDNA4 / HisMax-TOPO-Dp71A78 79 for the expression of the recombinant protein Xpress-Dp71A78 7g. B) Vector pcDNA3.1 / V5-His-TOPO-Dp71A78.79 for the expression of the recombinant protein Dp71A78 79-V5. The characteristics of each of the vectors are indicated. Taken from the manual of each of the vectors (Invitrogen) with some modifications.

Figure 6. Analysis of DH5a clones positive to Dp71A78.7g. Plasmid DNA was isolated from the candidates and analyzed by PCR assays. A) PCR product of the candidates selected for the vector pcDNA4 / HisMax-TOPO-Dp71A78 79 using the oligonucleotides XpF and rTAGDp71AR. B) PCR product of the candidates selected for the vector pcDNA3.1 / V5-His-TOPO-Dp71A78.79 using the oligonucleotides rATGDp71 F and BGH-R. The molecular weight markers (bp) are indicated in the left part of the figure. The arrow indicates the expected product. The box indicates the candidates selected for the sequencing. The PCR products were analyzed in 1.5% agarose gel stained with ethidium bromide.

Figure 7. Sequence of Dp71A78.79 cloned in vectors pcDNA4 / HisMax-TOPO- TA and pcDNA3.1A / 5-His-TOPO-TA. The sequence of the? 71? 78_79 cloned in the vectors was sequenced and compared to the sequence of the rat Dp71 a reported in the GenBank (NM_012698). A) Sequence of Dp71A78_79 obtained from the automatic sequencing of the vector pcDNA4 / HisMax-TOPO-Dp71A78 79, sequence identified as SEC. ID. No. 1 B) Alignment of exon 74 of Dp71 a with the sequence obtained from the sequencing of Dp71A78_79 corresponding to the same exon. C) Alignment of the amino acid sequence of exon 74 of Dp71a and Dp71A78 79. The alignment of the sequences was performed using the ClustalX program. The start and stop codons are indicated in bold. The box indicates the change and the codon which does not change the amino acid.

Figure 8. Expression of the vectors pcDNA4 / HisMax-TOPO-Dp71A78-79 and pcDNA3.1 / V5- His-Dp71A78.79. Transient transfection assays were performed on the cells HeLa and protein extracts were analyzed by WB assays. A) WB of extracts of HeLa cells transfected with the vector pcDNA4 / HisMax-TOPO-Dp71A78 79 using the anti-Xpress antibody. B) WB of extracts of HeLa cells transfected with the vector pcDNA3.1A / 5-Hys-TOPO-? 71? 78 79 using the anti-V5 antibody. Molecular weight markers (kDa) are indicated on the left side of each figure. The arrow indicates the recombinant protein detected. 1: non-transfected HeLa cells, 2 and 3: transfected HeLa cells. The β-actin protein used as load control is shown and was detected with the anti-actin antibody.

Figure 9. Characterization of PC12 cells transfected stably with plasmid pcDNA4 / HisMax-TOPO-Dp71A78 79. Zeocin-resistant clones (stable transfectants) were analyzed by genomic DNA PCR and by RT-PCR assays. A) Genomic DNA isolated from stable transfectants. B) PCR product of the genomic DNA of the stable transfectants using the oligonucleotides XpF and rTAGDp71AR. C) RNA isolated from stable transfectants. D) RT-PCR product of the stable transfectants using the XpF and rTAGDp71AR oligonucleotides. The molecular weight markers (bp) are indicated in the left part of figures B and D. The arrow indicates the expected product. The RT-PCR product of β-actin used as charge control is shown. Genomic DNA was analyzed in 0.8% agarose gel, RNA and PCR products in 1.5% agarose gel stained with ethidium bromide. C: clone.

Figure 10. Expression of the recombinant protein Xpress-Dp71A78-7g in the selected clones. Protein extracts were obtained from the stable transfectants and analyzed by WB assays. A) WB of the extracts of the selected clones using the anti-Xpress antibody. B) WB of the extracts of the clone C11 during the differentiation induced with NGF using the anti-Xpress antibody. Molecular weight markers (kDa) are indicated on the left side of each figure. It is indicated to the detected recombinant protein. The β-actin protein used as load control is shown and was detected with the anti-β-actin antibody. C1 1: cells PC12-C1 1, d: days.

Figure n. Analysis of the differentiation of cells PC12 and PC12-C1 1 induced with NGF. Photographs of the differentiated PC12 and PC12-C1 1 cells were taken and the percentage of differentiation and the average neurite length were determined. A) Non-differentiated PC12 cells. B) PC12 cells differentiated at 3 days. C) PC12 cells differentiated at 6 days. D) Non-differentiated PC12-C1 1 cells. E) Cells PC12-C1 1 differentiated to 3 days. F) PC12-C1 1 cells differentiated at 6 days. G) Percentage of differentiation calculated as described in example 1. H) Average of neurite length obtained as described in the examples. The graphs represent the average of three independent experiments ± the standard deviation. * P < 0.05, ** P < 0.01 and *** P < 0.001 indicate statistically significant differences. The scale is equal to 100 μ? T ?.

Figure 12. Differentiation of PC12 and PC12-C1 cells 1 in the absence of NGF. Photographs of PC12 and PC12-C1 1 cells cultured with differentiation medium without NGF were taken. A) Non-differentiated PC12 cells. B) PC12 cells cultured for 3 days. C) PC12 cells cultured for 6 days. D) Non-differentiated PC12-C1 1 cells. E) PC12-C1 1 cells cultured for 3 days. F) PC12-C1 1 cells cultured for 6 days. The scale is equal to 100 p.m.

Figure 13. Expression of the proteins Dp71a, Dp71e, Dp71 ab / Up71 and Up400 during the differentiation of PC12 and PC12-C11 cells. Protein extracts were obtained from PC12 and PC12-C1 1 cells and analyzed by WB assays. A) WB of the extracts of PC12 and PC12-C11 cells using the specific antibodies for each protein indicated in Table 1. B) Relative expression of Dp71a. C) Relative expression of Dp71e. D) Relative expression of Dp71ab / Up71. Molecular weight markers (kDa) are indicated in the left part of figure A. The arrow indicates the protein detected. The β-actin protein used as load control is shown and was detected with the anti-actin antibody. C1 1: cells PC12-C1 1, d: days. The graphs represent the average of three independent experiments ± the standard deviation. * P < 0.05, ** P < 0.01 and *** P < 0.001 indicate statistically significant differences.

Figure 14. Expression of Dp71 ab in cells PC12, PC12-D5 and PC12-D6. Protein extracts were obtained from PC12, PC12-D5 and PC12-D6 cells and analyzed by WB assays. A) WB of the extracts of cells PC12, PC12-D5 and PC12-D6 using the specific antibodies for each protein indicated in table 1. B) Relative expression of Dp71 a. C) Relative expression of Dp71 ab. The molecular weight markers (kDa) are indicated in the left part of figure A. The arrow indicates the protein detected. The β-actin protein used as load control is shown and was detected with the anti-actin antibody. D5 and D6: isolated colonies of stably transfected PC12 cells. The graphs represent the average of three independent experiments ± the standard deviation. ** P < 0.01 and *** P < 0.001 indicate statistically significant differences compared to PC12 cells.

Figure 15. Expression of ß-dystroglycan and Grb2 proteins during the differentiation of cells PC12 and PC12-C1 1. Protein extracts were obtained from cells PC12 and PC12-C1 1 and analyzed by WB assays. A) WB of the extracts of PC12 and PC12-C11 cells using the specific antibodies for each protein indicated in Table 1. B) Relative expression of β-dystroglycan. The molecular weight markers (kDa) are indicated in the left part of figure A. The arrow indicates the protein detected. The β-actin protein used as load control is shown and was detected with the anti-^ -actin antibody. C1 1: cells PC12-C 1, d: days. The graphs represent the average of three independent experiments ± the standard deviation. * P < 0.05 and *** P < 0.001 indicate statistically significant differences.

Figure 16. Expression of the NSE protein during the differentiation of PC12 cells and PC12-C1 1. Protein extracts were obtained from cells PC12 and PC12-C1 1 and analyzed by WB assays. A) WB of the extracts of PC12 and PC12-C11 cells using the anti-enolase antibody (H-300). B) Relative expression of NSE. The molecular weight markers (kDa) are indicated in the left part of figure A. The arrow indicates the protein detected. The β-actin protein used as load control is shown and was detected with the anti-^ -actin antibody. C11: PC12-C11 cells, d: days. The graphs represent the average of three independent experiments ± the standard deviation. ** P < 0.01 and *** P < 0.001 indicate statistically significant differences.

Figure 17. Subcellular localization of Dp71A78-79, ß-dystroglycan and Grb2 in cells PC12- C1 1 undifferentiated and differentiated with NGF. Indirect immunofluorescence assays were performed to determine the subcellular localization of each protein. A-C) and G-1) Subcellular localization of Dp7 A78-79, β-dystroglycan and Grb2 in undifferentiated PC12-C1 1 cells. PC12-C1 1 cells were immunostained A and G) with anti-Xpress antibody directed against recombinant Dp71A78-79, B) LG5 directed against β-dystroglycan or H) C-23 directed against Grb2. D-F) and J-L) Subcellular localization of Dp71A78.79, ß-dystroglycan and Grb2 in PC12-C11 cells differentiated at 6 days. PC12-C1 1 cells were immunostained D and J) with the anti-Xpress antibody directed against the recombinant Dp71A78 7g, E) LG5 directed against ß-distroglycan or K) C-23 directed against Grb2. C, F, I, L shows the overlap of the two previous images of each panel. The cells were analyzed using the TCS SPE confocal microscope and an equatorial cut was chosen to show the subcellular localization of each protein. The nuclei were stained with DAPI. The scale is equal to 10 μ? T? (C, I) and 25 μ ?? (F, L).

Figure 18. Subcellular localization of the recombinant proteins Xpress-Dp71A78-7gDp71A78-79-V5 in PC12 cells. Transient transfections of PC12 cells were performed with the vectors pcDNA4 / HisMax-TOPO-Dp71A78 79 and pcDNA3.1 / V5-His-TOPO-Dp71A78 79 and analyzed by indirect immunofluorescence to determine the subcellular localization of the recombinant proteins. A and D) Subcellular localization of Dp71A78 7g in cells PC 2. The transfected PC12 cells were immunostained A) with the anti-Xpress antibody directed against the recombinant protein Xpress-Dp71A78 79 or D) anti-V5 directed against the recombinant protein Dp71A78.79-V5. B and E) Nuclei stained with DAPI. C, F shows the over-position of the two previous images of each panel. The cells were analyzed using the TCS SPE confocal microscope and an equatorial cut was chosen to show the subcellular localization of each protein. The scale is equal to 10 μ ??.

Figure 19. Subcellular localization of the Xpress-Dp71A78-79 protein in MDCK cells.

Transient transfections of MDCK cells were performed with the vector pcDNA4 / HisMax-TOPO-Dp71A78 79 and analyzed by indirect immunofluorescence to determine the subcellular localization of Dp71A78 79. A and D) Subcellular localization of? 71? 78 79 in MDCK cells immunostained with anti-Xpress antibody directed against the recombinant protein Xpress-Dp71A78.79. B and E) Nuclei stained with DAPI. C, F shows the over-position of the two previous images of each panel. The cells were analyzed using the TCS SPE confocal microscope and an equatorial cut was chosen to show the subcellular localization of the Xpres-Dp71A78 7g protein. The scale is equal to 0 μ? T? (C) and 25 μ? T? (F).

Detailed description of the invention.

The present invention relates to nucleotide molecules, methods and compositions that stimulate neuronal differentiation.

The present invention shows the implication of the protein Dystrophin? 71? 78_79 in the process of neuronal differentiation, providing the bases for the development of new methods with therapeutic potential where the generation and / or stimulation of said neuronal differentiation is important. On the other hand, the present invention also relates to the use of Dp71A78 7g dystrophin to stimulate neuronal differentiation in cell lines, stem cells in general and neural stem cells in particular, in animal models or in individuals, particularly cell lines such as PC12. and in humans that present damage to the functioning of the nervous system and / or a certain degree of mental retardation caused, among other causes, by traumatism or diseases where there is damage or neuronal degeneration, such as Duchenne Muscular Dystrophy (DMD).

To achieve the above, in the present invention the nucleotide sequence coding for the mutant ?? 71? 78 79 was generated in vitro, this sequence being cloned into a molecular vector and resulting, for example, in the expression vector called pcDNA4 / HisMax -TOPO-TA- Dp71A78 7g, which is expressed stably in the cell line PC12 and consequently stimulates neuronal differentiation.

In one of its embodiments, the present invention provides nucleotide sequences encoding the Dp71A78 79 protein where said sequences lack the exons 71, 78 and 79 but conserve exons 1 and 63 to 70 and 72 to 77 of the dystrophin gene, including all those nucleotide sequences corresponding to exons of the DMD gene similar in at least 10%, capable of encoding said protein , wild and / or mutant, regardless of their origin, such as human, rat, mouse, etc., that are found in organisms in natural form or generated in vitro, including those proteins that have the function of stimulating neuronal differentiation in any system as well as the nucletidic sequences that encode them.

In another of its embodiments, the present invention provides vehicles or molecular vectors expressing sequences of the DMD gene, particularly coding for the protein Dp71A78 79, such as for example pcDNA4 / HisMax-TOPO-Dp71A78.79 and pcDNA3.1A 5-His- TOPO-Dp71A78 7g which express the protein Dp71A78 7g and which has the function of stimulating neuronal differentiation.

In another of its embodiments, the present invention provides compositions that can be of the pharmaceutical type, comprising a nucleotide sequence coding for the D671A78 79 Dystrophin protein or any nucleotide sequence of the DMD gene or variants, which upon stimulation stimulates neuronal differentiation, or either any protein product expressed from sequences of the DMD gene that has the function of stimulating neuronal differentiation.

The present invention also relates to the use of the nucleotide sequence or a sequence similar to this, which codes for the protein Dp71A78_79 dystrophin or for any product of the DMD gene or variants thereof, to prepare a medicament for stimulating neuronal differentiation in subjects who they present a certain degree of brain damage and / or mental retardation caused by trauma or by diseases where there is neuronal damage or neuronal degeneration as in the case of DMD.

In another of its embodiments, the present invention provides and describes the use of PC 12 cells transfected with a molecular vector containing the coding sequence for the Dp71A78.79 protein, or any sequence of the DMD gene; as a model or system of experimental study to stimulate the process of neuronal differentiation, also encompassing the use of other cell lines for the same purpose.

The level change in the expression of the different isoforms of Dp71 and the increase in the expression of β-dystroglycan and enolase are also described in the present invention, each of these proteins being able to be, or all of them together, the cause of the stimulation of neuronal differentiation observed in the present invention.

According to the present invention and in order to analyze the function of the dp71 domains, the Dp71A78 79 protein was generated as a mutant of the rat Dp71, through the in vitro elimination of exons 78 and 79 in its coding sequence (Figure 2), the resulting sequence being cloned into the expression vector pcDNA4 / HisMax-TOPO-TA (Figure 5A). As can be seen, the coding sequence for the protein? 71? 78. 79 lacks exons 78 and 79 of the COOH-terminal end, that is, it comprises the exon 1 of Dp71 and exons 63 to 77 of the DMD gene, with absence of exon 71 because in rat none of the isoforms of Dp71, identified so far, present exon 7159. Also, in the protein Dp71A78 79, no alters none of the interaction domains with the DAPs complex (Figure 2A) 129,130, however, the absence of exons 78 and 79 could modify the characteristics of said protein such as the effect on the neural differentiation of PC12 cells, their location subcellular, post-translational modifications and / or its folding. The function of Dp71A78 79 may be different from that of the Dp71 isoforms reported, allowing the function of exons 1 and 63 to be elucidated. 77 as well as the effect of the absence of exons 78 and 79 of Dp71.

To analyze the function of the protein? 71? 78 79, stably transfected PC12 cells were generated using the pcDNA4 / HisMax-TOPO-Dp71A78.79. Vector, observing that the PC12 cells stably expressing the Dp71A78 7g protein (PC12-C11) accelerate the differentiation process compared to non-transfected cells.

The present invention shows for the first time, that the expression of exons 1 and 63 to 77 and the absence of exons 78 and 79 of the DMD gene (gene coding for Dp71A78 79), stimulates the process of neuronal differentiation of PC12 cells, regulates the expression of the endogenous isoforms of Dp71, as well as the expression of ß-dystroglycan and neuron-specific enolase (NSE), which makes it possible to use the protein Dp71A78.7g as an active ingredient to stimulate neuronal differentiation in diseases where there is damage to the nervous system, such as in DMD.

For purposes of the invention, the protein Dp71A78 79 or proteins homologous to it, can be obtained by means of production methods of recombinant proteins known in the art, from host cells transformed with molecular vehicles containing sequences coding for said proteins.

Once the protein Dp71A78 79 of the present invention is obtained, it can be mixed with various elements that allow obtaining compositions pharmaceutical in various presentations that are convenient for administration according to what is previously known in the art, such as for example tablets, capsules, gels, solutions, suspensions, mouthwashes, granules, elixirs, emulsions, syrups, dispersions and other presentations comprising one or more additional factors that allow its administration and / or therapeutic effect and that are pharmaceutically acceptable as carriers.

On the other hand, the compositions of the invention can be administered as appropriate and by various routes, such as for example oral, intravenous, intramuscular, nasal or other routes, with parenteral routes such as intravenous and / or intramuscular being preferred.; Likewise, the dosage regimen can be extended as necessary, including daily administrations until adequate treatment is provided, according to the weight, age and condition of the patient.

For parenteral administration, the compositions of the invention comprise sterile injectable preparations of the active ingredient, for example the protein 71 71 78, in a solution preferably isotonic and compatible with the blood of the subject to be treated. Said compositions comprise aqueous and / or non-aqueous formulations which may contain convenient adjuvants, such as buffers, bacteriostats, sugars and the like; likewise said compositions can be presented in single dose form or in multi-dose containers, for example sealed ampules or vials.

The compositions described herein can be prepared according to methods known in the art, which include the association of the protein Dp71A78.79 with a pharmaceutical carrier consisting, for example, of several ingredients. In general, said compositions are prepared in such a way that the? 71? 78_79 protein of the invention and the rest of its components are associated in a homogenous and intimate way to generate the pharmaceutical form that is of interest.

In the case of compositions containing the Dp71A78_79 protein that can be applied topically, the carriers that can be used for this purpose include those that are topically acceptable, and included in various presentations such as, for example, drops, colines, aerosols, sprays, lotions, gels, creams, ointments, liposomes or similar.

According to the present invention, the protein? 71? 78 79 can be employed to provide a method of treating mammals with conditions where nervous system damage has been caused, such as in DMD, which comprises administering to the mammalian subject an amount of the protein Dp71A78_7g in a pharmaceutically acceptable form and in therapeutic quantities such as to allow treating said condition.

Therapeutic protocols that can be carried out to use the Dp71A78 7g Dystrophin nucleic acid, the vector containing it or the protein encoded or expressed by this nucleic acid molecule are well known to any expert with average knowledge in the area: There are several documents that describe these therapeutic protocols in detail, such as, for example, the protocols described in: Molecular Cloning: A Laboratory Manual, 2nd Ed., Ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989); AD Miller Progress towards human gene therapy. fí / ooc 76: 271-278, 1990; Current Protocols in Molecular Biology, Ausubel, F.M. et al., (Eds.) Greene Publishing Associates, (1989), W019900 1092, US5679647, more specifically the protocols described for treating muscular dystrophies and which are described in EP1423133 and US2010 / 0292306.

As will be seen in the following examples, the present invention allows to provide suitable therapies to subjects suffering from damage to the nervous system caused by pathological conditions, such as DMD, for example, by administering compositions comprising the protein Dp71A78 7g, which stimulates neuronal differentiation in these subjects.

The following examples are shown below only for the purpose of illustrating the present invention and without implying any limitation in its scope.

Example 1. Materials and methods, a) Cell culture and differentiation.

PC12 cells were cultured in plastic P100 boxes with DMEM growth medium (Gibco) supplemented with 10% horse serum (Gibco), 5% fetal serum from bovine (Gibco), 100 U / ml penicillin (Gibco), 1 mg / ml streptomycin (Gibco) and 0.25pg / ml mycostatin (Gibco) at 37 ° C in a water saturated atmosphere with 5% CG- 2.

For differentiation, PC12 cells were seeded with synchronization medium (diluted growth medium 1: 10) at a density of 60-80% confluence in plastic boxes P60 or P100 pre-treated with collagen for 30 minutes or on treated coverslips. with Poly-L-Lysine. The cells were incubated at 37 ° C for 72 hours in a water saturated atmosphere with 5% CO2. Then, the medium was replaced by means of differentiation (synchronization medium with 50 ng / ml of NGF 2.5S, Invitrogen). The cells were harvested at 0, 1, 3 and 6 days of differentiation to obtain total protein extract or analyzed by indirect immunofluorescence. The growth medium was changed twice a week and the means of differentiation every three days.

The HeLa and MDCK cell lines were grown in plastic P100 boxes with DMEM growth medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) for HeLa and 5% for MDCK, 100 U / ml penicillin (Gibco) , 1 mg / ml of streptomycin (Gibco), 0.25 pg / ml of mycostatin (Gibco) and 1 mM of sodium pyruvate at 37 ° C in a water-saturated atmosphere with 5% CO2. The growth medium was changed twice a week. b) Total RNA extraction.

The RNA was obtained from cell cultures grown at 90% confluence in the case of non-transfected PC12 and undifferentiated transfected PC12 cells. Isolation of total RNA was carried out using the commercial reagent Trizol (Gibco), following the supplier's instructions. The RNA pellet was resuspended in 50 μ? of water treated with diethyl-pyrocarbonate (DEPC). The total RNA concentration was obtained in a spectrophotometer and 1 μg of total RNA was mixed with 1 μ? sample buffer BXG 6X (bromophenol blue 0.25%, xylene cyanol 0.25%, glycerol 30%). Subsequently, the integrity of the RNA was verified by electrophoresis in 1.5% agarose gels (Gibco) in 0.5X TBE buffer and stained with ethidium bromide. The RNA was stored at -70 ° C. c) Reverse transcription coupled to PCR (RT-PCR).

For the synthesis of the cDNA, the RNA was treated with DNasal (Invitrogen) to eliminate the contamination with genomic DNA. The "SuperScript III First-Strand Synthesis for RT-PCR" kit (Invitrogen) was used, for which a mixture of 20 μ? in a 0.2 ml sterile PCR tube with the following components: 1 μ? of oligonucleotide specific for dystrophin (dcDNA) (table 1) or 1 μ? of oligonucleotide dT, 3 μg of total RNA free of DNA, 1 μ? of dNTP mix (10 mM each) and the required volume of water with DEPC to complete a final volume of 10 μ ?. This mixture was incubated at 65 ° C for 5 minutes and then placed on ice for two minutes, after which 10 μ? Was added to this tube. of a pre-mix of 2 μ? buffer RT 10X, 4 μl of 25 mM MgCl2, 2 μ? of DTT 0.1 M, 1 μ? of RNase OUT 40 U / μ? and 1 μ? of SuperScript III RT 200 U / μ ?. Subsequently, this mixture was incubated at 55 ° C for 60 minutes followed by an incubation for 5 minutes at 85 ° C. Finally, 1 μ? Was added to the reaction? of RNase H and incubated at 37 ° C for 20 minutes. The final reaction was stored at -20 ° C until its use.

The analysis of the quality of the cDNA was carried out by means of the amplification of the ß-actin mRNA by the PCR technique using specific oligonucleotides, for which the following components were mixed: 5 μ? of PCR buffer 10X, 1.5 μ? of 50 mM MgCl 2, 1 μ? of dNTP mix (10 mM each), 1 μ? of oligonucleotide Actin 1 (200 ng / μ?), 1 μ? of oligonucleotide Actin 2 (200 ng / μ?), 2 μ? of the product of retro-transcription (RT) with oligo dT (cDNA), 0.3 μ? of Taq DNA Polymerase (2 U / μ?) and the required volume of sterile MQ water for 50 μ ?.

The PCR reaction was performed in an Applied Biosystems thermocycler (Gene Amp PCR System 2700). The transcript of Dp71A78.7g was amplified using the methodology previously described with the oligonucleotides rATGDp71 F and rTAGDp7lAR and 2 μ? of the product of the RT with the oligo dcDNA (oligo specific for the mRNAs of the dystrophins). The first oligonucleotide aligns with the sequence of exon 1 of Dp71 (AUG) and the second is complementary to the last region of exon 77 of the DMD gene; the last oligo has the stop codon UAG to finish the translation of the protein. This pair of oligonucleotides allowed the amplification of the cDNA of Dp71A78.79.

On the other hand, to identify the transcript of the recombinant protein Xpress-Dp71A78.79 in the stable transfectants, the oligonucleotides "Xpress Forward" (XpF), specific for the vector, and rTAGDp71AR with 2 μ? Were used. of the RT generated with the oligo dT. HE analyzed 5-10 μ? of each PCR reaction mixed with 1 μ? sample buffer BXG 6X (bromophenol blue 0.25%, xylene cyanol 0.25%, glycerol 30%) in 1.5% agarose gels in 0.5X TBE buffer and stained with ethidium bromide.

The sequence of all the oligonucleotides used in the present invention, PCR conditions and the size of the amplified products are shown in Table 1. d) Construction of the vectors pcDNA4 / HisMax-TOPO-Dp71A78_79 and pcDNA3.1 V5-His-TOPO-Dp71A78 79.

- Vector pcDNA4 / HisMax-TOPO-Dp71A78.79.

The vector pcDNA4 / HisMax-TOPO-TA (figure 5A) was used to fuse the epitope "Xpress" to the NH2-terminal region of Dp71A78_79. The vector pcDNA4 / HisMax-TOPO-TA contains the CMV promoter from which the recombinant protein Xpress-?? 71? 78 79 and the zeocin resistance gene are expressed to select stable transfectants from PC12 cells. For the construction of the vector pcDNA4 / HisMax-TOPO-Dp71A78.79, the sequence of Dp71A78 79 was amplified by PCR from 2 μ? of cDNA from cells PC12 using the high fidelity Taq "AccuPrime Pfx DNA Polymerase" (Invitrogen) with the oligonucleotides rATGDp71 F and rTAGDp71AR and following the instructions of the Taq recommended by the supplier. The amplified product was ligated into the vector pcDNA4 / HisMax-TOPO-TA according to the supplier's instructions (Invitrogen). The ligation mixture was used to transform E. coli strain DH5a. The colonies that were obtained were analyzed by PCR with the oligonucleotides XpF and rTAGDp7lAR. One of the plasmids was sequenced using the "Dye deoxy terminator cycle sequence" kit with the "ABI Prism sequencing apparatus 310" (Applied Biosystems). The plasmid concentrations and integrity were analyzed by means of electrophoresis in 1% agarose gels and visualized by staining with ethidium bromide.

- Vector pcDNA3.1A 5-His-TOPO-Dp71A78 79.

The vector pcDNA3.1 / His-TOPO-TA (Figure 5B) was used to fuse the epitope "V5" to the COOH-terminal region of Dp71A78 79. The vector pcDNA3.1 / His-TOPO-TA contains the CMV promoter to regulate the expression of the recombinant protein Dp71A78.79-V5.

Table 1. Oligonucleotide sequence, product size and PCR conditions.

The sequence of the oligonucleotides was designed based on the mRNA of rat Dp71 reported in GenBank (NM 012698). The sequence of the oligonucleotides XpF and BGH-R were taken from the manual of the vector pcDNA4 / HisMax-TOPO-TA (Invitrogen). Actin 1 and Actin 2 are sense and antisense oligonucleotides, respectively.

For the construction of this vector, the sequence of Dp71A78_79 was amplified by PCR from the vector pcDNA4 / HisMax-TOPO-Dp71A78_79 using high fidelity Taq "AccuPrime Pfx DNA Polymerase" with the oligonucleotides rATGDp71 F and rDp71AR-V5. With the oligo rDp7lAR-V5, the stop codon was eliminated to allow fusion of the V5 flag to Dp71A78 79. The amplified product was purified from the agarose gel by electroelution131 and ligated into the vector pcDNA3.1 / His-TOPO- TA following the supplier's instructions (Invitrogen). The ligation reaction was used to transform E. coli strain DH5a. The colonies that were obtained were analyzed by PCR with the oligonucleotides rATGDp71 F and BGH Reverse (BGH-R), the latter specific for the vector. One of the plasmids was sequenced, as previously described, and compared to the sequence of? 71? 78 _79 in the vector pcDNA4 / HisMax-TOPO-Dp71A78 79. e) Transient transfection of HeLa, PC12 and MOCK cells.

- HeLa cells.

In order to determine the expression of the protein Xpress-Dp71A78 79 and Dp71A78,79-V5 from the vectors pcDNA4 / HisMax-TOPO-Dp71A78.79 and pcDNA3.1 / His-TOPO-Dp71A78.79, respectively, performed a transient transfection of HeLa cells. HeLa cells were seeded at 80% confluence in a P60 box 24 hours before transfection. For transfection, the following mixtures were prepared: in a 500 μ? Tube. of Opti-MEM (Gibco) with 5 g of the vector pcDNA4 / HisMax-TOPO-Dp71A78,79 or pcDNA3.1 / His-TOPO-Dp71A78.79 and in another tube 500 μ? of Opti-MEM with 5 μ? from Lipofectamine 2000 (Invitrogen). The mixtures were incubated for 10 minutes at room temperature (RT), then the components of both tubes were mixed and the mixture was incubated again at RT for 40 minutes for the formation of the transfection complexes. To the DNA-Lipofectamine 2000 mixture was added 2 ml of Opti-MEM and incubated again for 10 minutes at RT. Before 10 minutes, the cells were washed twice with Opti-MEM and the transfection medium was added. The cells with the transfection medium were incubated for 5 hours at 37CC in a saturated water atmosphere with 5% CO2. Finally, to the transfected cells, 2 ml of growth media were added. The cells were harvested 24 hours post- transfection and the expression of the protein Dp71A78.79 was analyzed by Western Blot (WB).

- PC12 cells.

PC12 cells were seeded at 90% confluence with synchronization media on coverslips previously treated with Poly-L-Lysine. After 24 hours of sowing the transfection was carried out as follows: in one tube, 100 μl of Opti-MEM (Gibco) was mixed with 2 pg of the vector pcDNA4 / HisMax-TOPO-Dp71A78.79 or pcDNA3.1 / His-TOPO- ?? 71? 78.79 and in another tube 100 pl of Opti-MEM with 20 μ? of Lipofectamine 2000 (Invitrogen). The preparations were incubated for 10 minutes at RT, then the components of both tubes were mixed and incubated at RT for 40 minutes for the formation of the transfection complexes. To the DNA-Lipofectamine 2000 mixture was added 800 μ? of Opti-MEM and incubated again for 10 minutes at RT. Meanwhile, the cells were washed twice with Opti-MEM, the transfection medium was added and they were incubated for 5 hours at 37 ° C in a water saturated atmosphere with 5% CO2. Finally, 1 ml of growth medium was added to the transfected cells. The transfected cells were analyzed by immunofluorescence 24 hours post-transfection.

- MDCK cells.

The MDCK cells were seeded at 80% confluence on coverslips previously treated with Poly-L-Lysine 24 hours before transfection. The transfection was carried out by mixing 100 μ? In a tube. of Opti-MEM (Gibco) with 2 pg of the vector pcDNA4 / HisMax-TOPO-Dp71A78.79 and in another tube 100 μ? of Opti-MEM with 4 μ? of Lipofectamine 2000 (Invitrogen). The preparations were incubated for 10 minutes at RT, then the components of both tubes were mixed and incubated at RT for 40 minutes for the formation of the transfection complexes. Once the DNA-Lipofectamine 2000 transfection complexes were formed, 800 μ? of Opti-MEM and incubated again for 10 minutes at RT. Before 10 minutes, the cells were washed twice with Opti-MEM and the transfection medium was added. The cells with the transfection medium were incubated for 5 hours at 37 ° C in a water saturated atmosphere with 5% CO2 and then 1 ml of growth medium was added. The cells Transfected cells were analyzed by immunofluorescence after 24 hours of transfection. f) Stable transfection of PC12 cells.

To generate stable transfectants of PC12 cells with Dp71A78 79, the cells PC12 were seeded at 90% confluence with synchronization medium in P100 boxes treated with collagen. After 24 hours of gluing, the transfection mixture was prepared, on the one hand by mixing 800 μ? of Opti-MEM (Gibco) with 15 μ of the vector pcDNA4 / HisMax-TOPO-Dp71A78.79 and on the other hand 800 μ? of Opti-MEM with 50 μ? of Lipofectamine 2000 (Invitrogen), following the incubation times described above. To the mixture of the transfection complexes, 4.5 ml of Opti-MEM was added and incubated for 10 minutes at RT. The cells were washed twice with Opti-MEM and the transfection medium was added. After 5 hours of incubation with the transfection medium, 6 ml of growth medium was added. The transfection medium was replaced by means of selection, consisting of growth medium added with zeocin (Invitrogen) at a final concentration of 400 pg / ml, and incubated 48 hours post-transfection. Approximately six months later, zeocin-resistant colonies were isolated. Clones were obtained from one of the colonies by serial dilution in 96-well boxes. The clones that were obtained (figure 9) were characterized by genomic PCR, RT-PCR and WB to determine stable transfection. Stably transfected cells were maintained with the growth medium added with Zeocin at 200 μS / G. g) Extraction of genomic DNA.

The cells from the clones were removed from the boxes, recovered by centrifugation and washed with PBS (15 mM KH2PO4)., 15 mM K2HPO4, 154 mM NaCl2, pH 7.2). The cells were resuspended in 200 μ? of lysis buffer (Tris-HCI 500 mM pH 8, EDTA 00 mM pH 8.0 and Sarcosyl 0.5%) added with RNase A at a final concentration of 100 ng / ml. The mixture was incubated at 37 ° C for 1 hour and then Proteinase K was added to a final concentration of 100 pg / ml and incubated at 65 ° C for 1 hour. Subsequently, it was mixed by inversion with a volume of phenol saturated with Tris-HCI pH 8 and centrifuged at 12,000 rpm for 2 minutes. The aqueous phase was removed and re-mixed with phenol until the cell debris was removed. The aqueous phase was recovered, mixed with a volume of chloroform and centrifuged at 12,000 rpm for 2 minutes. Finally, to the aqueous phase was added half a volume of 7.5 M ammonium acetate and 2 volumes of cold absolute ethanol and the DNA was allowed to precipitate at -20 ° C overnight. The next day we proceeded to centrifuge at 12,000 rpm for 15 minutes. The DNA pellet was resuspended in 50 μ? of water treated with DEPC and the total concentration was obtained in a spectrophotometer. 1 pg of total DNA was mixed with 1 pl of BXG 6X sample buffer and its integrity was verified by electrophoresis in 0.8% agarose gels (Gibco) in 0.5X TBE buffer and stained with ethidium bromide. The DNA extract was stored at -20 ° C. h) Preparation of protein extract.

The cells were removed from the boxes, recovered by centrifugation and washed with PBS. Subsequently, the cells were resuspended in 200 μ? of extraction buffer A (250 mM Tris-1 mM EDTA, pH 8.0) with COMPLETE protease inhibitors (Roche, Inc.). The cells were lysed by sonication and the concentration of the protein was determined by the Bradford method. The total protein extract was resuspended in electrophoresis buffer (Trís-HCI 75 mM, SDS 15%, β-mercaptoethanol 5%, glycerol 20%, bromophenol blue 0.001%). The protein extracts were stored at -20 ° C. i) Western blot.

Aliquots of 60-80 pg of total protein were prepared and subjected to electrophoresis in SDS polyacrylamide gels in a gradient of 4 to 12%. The proteins were transferred to nitrocellulose membranes for 18-20 hours at 70 mAmp and at a temperature of 4 ° C. The nitrocellulose membrane with the proteins was blocked with 5% skim milk in 1X TBS-T buffer (10 mM Tris pH 7.4, 150 mM NaCl, 0.05% Tween 20) for 2 hours under stirring at RT. Subsequently, the membrane was incubated overnight with the primary antibody for each protein diluted in TBS-T 1X buffer alone or with 5% skimmed milk. The antibodies used in the Western blot assays, type of antibody, recognizing protein and the dilution of each of them are shown in Table 2. The secondary antibodies were anti-mouse IgG or anti-rabbit IgG conjugated to peroxidase for the detection of the primary antibody at a dilution of 1: 10000 in blocking buffer. The immunoreactive bands were detected according to the Western-Blot ECL system (Amersham Pharmacia Biotech). As a molecular weight marker, 3.5 μ? from "Prestained SDS-PAGE Standard, Broad Range" (Bio-Rad).

Table 2. Antibodies used and their characteristics 1 Intentions of invitrogen; 2Donado by Dr. Dominique Mornet, France; 3 Obtained from Santa Cruz Biotechnology; "Produced by Washington Biotechnology Inc.

(Columbia, USA). 5Donated by Dr. Manuel Hernández (Department of Cell Biology-CINVESTAV, Mexico, D. F); IF: Indicates the immunofluorescence conditions. j) Indirect immunofluorescence assays.

The cells were grown on coverslips previously treated with Poly-L-lysine, transfected or differentiated as previously described. Coverslips were removed from the 6-well box and washed with cytoskeletal buffer (CB) (10mM MES, 150mM NaCl, 5mM EGTA, 5mM MgCl 2, 5mM Glucose). The cells were then permeabilized with 0.4% Triton X-100 and 4% paraformaldehyde in CB buffer for 5 minutes. Subsequently the cells were washed three times for 5 minutes with CB buffer and fixed with 4% paraformaldehyde in CB buffer for 20 minutes. After washing with PBS, nonspecific sites were blocked with 0.5% gelatin in PBS for 40 minutes. After this, the coverslips were washed with PBS and incubated overnight at 4 ° C with the corresponding primary antibody diluted in PBS. The next day the coverslips were washed with PBS to remove excess antibody and incubated for 1 hour at RT with secondary antibody Alexa488 or Alexa596 (Amersham life science products) for monoclonal or polyclonal antibodies, respectively, diluted in PBS.

Finally, coverslips were washed with PBS and mounted on slides with Vectashieid (Vector laboratories, Inc.). The preparations were analyzed with the Leica TCS SPE confocal microscope using the 63x objective. For each of the images, 20-25 cuts were taken at intervals of 0.3 μm and the equatorial cut was chosen to show the subcellular localization of the proteins analyzed. k) Quantitative analysis of cell differentiation.

The comparison of the differentiation between the non-transfected PC12 cells and the transfected PC12 was carried out in the following way: photographs were taken, in an inverted microscope, to 10 fields chosen at random from each of the cells boxes at 0, 3 , 6 and 9 days of differentiation.

To determine the percentage of differentiation, the absence and / or presence of neurites were taken into account.

Cells with neurites were counted manually and the percentage of differentiation was obtained with respect to the total number of cells of the 10 fields for each of the days analyzed. Subsequently, the length of the neurites of the differentiated cells was measured using the AxioVision LE Reí program. 4.7 (Zeiss). Once the measurements were made, we proceeded to obtain the average of the neurite length of the differentiated cells in each of the days analyzed.

I) Statistical analyzes.

The values shown in the figures are the mean ± the standard deviation obtained from three independent experiments.

The data were analyzed in "unpaired Student's t-test" with the GraphPad Prism 5 program. The value of P used as statistical difference criterion was a value less than 0.05 (P <0.05).

Example 2. Construction of vectors pcDNA4 / HisMax-TOPO-Dp71A78 79 and pcDNA3.1 / V5-His-TOPO-Dp71A78_79 Due to the absence of specific antibodies to distinguish each of the isoforms of Dp71 and in order to elucidate the function of the dp71 domains, the vectors pcDNA4 / HisMax-TOPO-Dp71A78.79 and pcDNA3.1 / V5-His-TOPO-?? 71? 78 79 with the sequence of Dp71A78 79 (FIGS. 5A and B, respectively) as described above. The transformant colonies that were obtained after transforming the E. coli strain DH5a with the vector pcDNA4 / HisMax-TOPO-Dp71A78 79 were analyzed by PCR using the oligonucleotides XpF and rTAGDp71AR (Table 1). Figure 6A shows the 1852 bp PCR product corresponding to the Xpress-Dp71A78 79 DNA amplified from the vector pcDNA4 / HisMax-TOPO-Dp71A78 79. To determine that the sequence of the Dp71A78 79 cloned in this vector is not had no change, candidate number 5 was sequenced and compared to the sequence of the rat Dp71 reported in the GenBank database (NM_012698). The sequence of the cDNA of Dp71A78 79, sequence identified as SEQ, is shown in Figure 7A. ID. No. 1. When comparing the results of the sequencing with the coding sequence of the rat Dp71, a change was found in position 1221 of T by C, which corresponds to exon 74 (FIG. 7B); however, this change does not affect the amino acid sequence (Figure 7C). The isoforms of Dp71 have an actin-binding domain in the NH2-terminal region83 and the epitope "Xpress" fused in this region could interfere with the function of Dp71A78 79. In order to rule out this possibility, the sequence of the Dp71A78.79 in the vector pcDNA3.1 / V5-H¡s-TOPO-TA, which adds the epitope "V5" in the COOH-terminal region of Dp71A78 79. The sequence of Dp71A78 79 was amplified by PCR to from the vector pcDNA4 / HisMax-TOPO-Dp71A78 7g and was cloned into the vector pcDNA3.1A / 5-His-TOPO-TA (Figure 5B) as described above. The candidates obtained for the vector pcDNA3.1 / V5-His-TOPO-Dp71A78 79 were analyzed by PCR.

In Figure 6B a 1948 bp band is observed which corresponds to the sequence cloned into the vector pcDNA3.1A / 5-His-TOPO-TA. Plasmid 6 was sequenced and the results of the sequencing were compared with the sequence of Dp71A78 79 cloned into the vector pcDNA4 / HisMax-TOPO-Dp71A78.79. No change in the sequence was found Dp71A78_79 cloned in pcDNA3.1 / V5-His-TOPO-TA with respect to the vector of origin. Both vectors were used to perform transient transfections and the vector pcDNA4 / HisMax-TOPO-Dp71A78.79 was used to generate stable transfectants of PC12 cells.

Example 3. Expression of the vectors pcDNA4 / HisMax-TOPO-Dp71A78 79 and pcDNA3.1 / V5- His-TOPO-Dp71A78 79 in HeLa cells.

To evaluate whether the expression vectors pcDNA4 / HisMax-TOPO-Dp71A78.79 and pcDNA3.1A / 5-His-TOPO-Dp71A78 79 correctly expressed the recombinant proteins Xpress-Dp71A78 79 and Dp71A78 79-V5 respectivelyTransient transfection tests were carried out for 24 hours in HeLa cells. The protein extracts of the transfected and non-transfected cells were analyzed by WB using specific antibodies for each of the epitopes (table 1). When carrying out the WB with the anti-Xpress antibody, it detects a protein below the 80 kDa marker, only in the transfected cells, and that corresponds to the expected molecular weight (Figure 8A). The proteins of the cells transfected with the vector pcDNA3.1 / V5-His-TOPO-Dp71A78.79 were analyzed with the anti-V5 antibody. The results obtained with this antibody are shown in Figure 8B where the expression of a protein of the molecular weight corresponding to Dp71A78 79-V5 is observed only in the transfected cells.

Example 4. Generation of stable transfectants of PC12 cells with plasmid pcDNA4 / HisMax-TOPO-Dp71A78 7g.

Once the functionality of the vector was corroborated, we proceeded to generate stably transfected cells with the pcDNA4 / HisMax-TOPO-Dp71A78.79 plasmid, in order to analyze the effect of the over-expression of the recombinant protein Xpress- Dp71A78 7Q in the differentiation of PC12 cells, as well as the subcellular localization of this protein. The transfection was performed as described above. The cells were cultured in selection medium (growth medium added with Zeocin) for several weeks until the control cells (untransfected) died. The cells that survived the selection time were isolated and kept in culture with selection medium. Subsequently, a serial dilution was performed in a 96-well box to obtain clones of the transfected cells. We were able to identify 10 clones of which 9 were analyzed by PCR of genomic DNA, to determine if the integration of the plasmid to the genome of the transfected clones had occurred. For this part of the work, the DNA of the selected clones was obtained, including the wild-type PC12 cells (Figures 9A); subsequently, the DNA was amplified by the PCR technique using the oligonucleotides XpF and rTAGDp71AR, specific for the cDNA of the recombinant protein Xpress-Dp71A78 79. Figure 9B shows the PCR products of 1852 bp corresponding to the expected molecular weight; this result confirmed that the selected clones had inserted the plasmid into the genomic DNA.

Stable transfectants were obtained with plasmids homologous to those previously described that contained the cDNA sequences of the Dp71 a, Dp71ab and Dp71c isoforms. These stable transfectants did not express the corresponding proteins, analyzed by WB. The only cells that expressed the protein were those transfected with Dp71A78 79. This result suggests that the endogenous proteins are subject to a regulation that does not allow overexpression.

Example 5. Characterization of the expression of the recombinant protein Xpress-Dp71A78 7g in the selected clones.

To determine whether stable transfectants expressed Dp71A78 79, the next step was to analyze the expression of messenger RNA (mRNA) of Dp71A78 79, for which the total RNA of the 10 clones was isolated (figure 9C) and tests were performed. of RT-PCR. Figure 9D shows that the 10 selected clones express the mRNA of the recombinant protein, but not the control cells (non-transfected PC12 cells); these results were obtained with the same oligonucleotides used in the analysis of genomic DNA. Once established that the selected clones expressed the mRNA of the recombinant protein, the analysis was continued at the protein level. Figure 10A shows a WB assay with the anti-Xpress antibody to analyze the expression of Dp71A78_7g, from the total protein extract of the transfected clones. The results indicate that all the selected clones express the recombinant protein Xpress-Dp71A78 7g. This immunoreactive band is absent in non-transfected PC12 cells. Clone 1 1 (PC12-C1 1) was selected to analyze the expression of Dp71A78 79 during differentiation with NGF. The protein extracts were obtained from the differentiated PC12-C1 1 cells at 1, 3 and 6 days and these were analyzed by WB. The results of Figure 10B show the expression of the Dp71A78 79 protein in all the days of differentiation analyzed.

Subsequently, indirect immunofluorescence assays were performed and the subcellular localization of Dp71A78 79 was determined by confocal microscopy as well as its colocalization with the β-dystroglycan and Grb2 proteins.

Example 6. Stimulation of the differentiation of clones PC12-C11.

Preliminary results to this work had shown that PC12 cells that stably expressed the? 71? 78_79 had a stimulation in the differentiation process with NGF. Nevertheless, these results were obtained from an isolated colony after a transfection experiment. In the present invention we decided to obtain clones of stable transfectants to work with a homogeneous lineage. From the clones selected and previously described, the clone C11 (PC12-C1 1) was selected for the quantitative analysis of the differentiation. By inducing differentiation with NGF of this selected clone, a stimulation was observed in the differentiation process of the PC12-C11 clones compared to the non-transfected PC12 cells (Figures 11A to 11F). These observations were quantified and analyzed as described above. When determining the percentage of differentiation, as well as the average neurite length, it was observed that PC12-C1 1 cells presented a differentiation percentage of 75.06% ± 4.37 on the first day, 84.13% ± 2.30 on the second day and 88.21% ± 2.38 on the third day of differentiation induced by NGF; these values are significantly higher than that of the non-transfected PC12 cells, which are 33.39% ± 3.17, 51.10% ± 2.97 and 61.87% ± 3.25 on the same days of treatment with NGF respectively (figures 1 1 G). The average length of neurites yielded the following results: PC12-C1 1 cells presented neurites with an average length of 16.36 ± 0.85, 29.77 ± 7.02 and 44.67 ± 6.19 pm at 1, 2 and 3 days of differentiation respectively; while non-transfected PC12 cells possess neurites with an average length of 8.96 ± 2.40, 13.44 ± 4.80 and 17.37 ± 6.80 pm at the same day differentiation (Figure 1 1 H). The results indicate that the transfected cells have a greater capacity to respond to NGF, generating a greater number of differentiated cells, as well as neurites of greater length in the first days of differentiation. Observations were made at different days of differentiation and phenotypically untransfected PC12 cells are completely differentiated between 9 to 12 days of treatment with NGF, while in PC12-C1 1 cells the same phenotype was observed between 4 to 6 days of treatment; however, the quantifications were not performed due to the complexity to identify the start and end of the neurites. In the present invention, the behavior between the PC12 and PC12-C1 1 cells cultured in differentiation media without complementing with NGF was also compared and it was observed that the PC12-C1 cells show an extension of short neurites, which are absent in untransfected PC12 cells, suggesting that PC12-Dp71A78 79 cells present a differentiation stimulation phenotype that is independent of NGF (Figure 12A-F).

Example 7. Analysis of the expression levels of Dp71a, Dp71e, Dp71ab / Up71 and Up400 in PC12-C11 cells.

The importance of the Dp71 isoforms in the differentiation of PC12 cells has been reported in several studies59,84,100,111. To try to explain the stimulation of the differentiation of the transfected cells, it was decided to determine the expression levels of the proteins Dp71a, Dp71e, Dp71ab / Up71 and Up400. Total protein extracts were isolated from PC12-C1 1 and PC12 cells and WB assays were performed using antibodies specific for each protein. Figure 13A shows that in undifferentiated PC12-C1 1 cells the amount of Dp71 a is decreased compared to untransfected PC12 cells. The decrease is maintained during the differentiation of PC12-C1 1 cells (Figure 13B); while in nontransfected cells it remains constant as previously described59.

Our working group has described the expression of a new isoform of Dp71 (Dp71e) in PC12 cells as described previously132. In Figure 13A and C it is shown that the expression of Dp71 e in PC12-C1 1 cells is decreased with respect to non-transfected PC12 cells in both undifferentiated cells as well as in differentiated cells. On the other hand, it is known that the absence of Dp71 increases the expression of the Up71 protein, a protein homologous to Dp7178,100, so that the decrease in Dp71 and the overexpression of Dp71A78 79 could be affecting the expression of the Up71. To test this hypothesis, we analyzed the expression of the Dp71ab / Up71 proteins using the H5A3 antibody directed against dystrophin and utrophin. The results of this experiment showed that the level of these proteins is increased both in undifferentiated PC12-C11 cells and in those differentiated with NGF with respect to non-transfected PC12 cells (Figure 13A and D). Because the H5A3 antibody recognizes both dystrophin and utrophin, these proteins may correspond to Dp71ab and / or Up71. Initially the expression of Dp71ab was analyzed in colony 5 (D5) from where the PC12-C11 cells were obtained, using the 5F3 antibody specific for this protein. In this colony, as in the D6 colony, an increase in the expression of Dp71ab was observed compared to the transfected cells (Figure 14A and 14C). In these same cells the decrease in the expression of Dp71a was also found (Figure 14A and 14B). On the other hand, the expression of utopofin Up400 between cells PC12 and PC12-C1 1 was compared using the K7 antibody, in which we did not find any change in the expression of this protein between the two cell types in both undifferentiated cells as in the differentiated ones (figure 13A). With these results, a differential regulation of the Dp71 and / or Up71 isoforms in the transfected cells was demonstrated.

Example 8. Expression of β-dystroglycan and Grb2 in PC12-C11 cells.

As part of the characterization of clone PC12-C1 1 it was decided to analyze the expression levels of β-dystroglycan and Grb2. Β-dystroglycan is a protein that is part of the DAPs / Dp71 complex in pci284 100'102 cells. Grb2 is an adapter protein of the signaling pathway related to Ras that forms a complex with β-dystroglycan68, 69.7 °. Figure 15A shows the levels of the β-dystroglycan protein in undifferentiated PC12 and PC12-C1 1 cells and throughout the differentiation induced by NGF. An increase in the expression of β-dystroglycan is observed in PC12-C1 cells not differentiated with respect to non-transfected PC12; this increase is maintained on days 1 and 3 of differentiation (figure 15B). The level of β-dystroglycan remains constant during the differentiation of untransfected PC12 cells (Figure 15A-B) as previously reported by Márquez et al59. When comparing the expression levels of the Grb2 protein between undifferentiated PC12 and PC12-C1 1 cells and during differentiation, no variation was found between the two cell types in both the undifferentiated cells and in the cells differentiated with NGF ( Figure 15A).

Example 9. Expression of NSE during the differentiation of PC12-C11 cells.

Neuron-specific enolase (NSE) is expressed mainly in nerve tissue and its expression is related to the process of differentiation and functional maturation of brain neurons133. NSE has also been described as a marker of neural differentiation of cells pci2134,135,136 and other neuronal cell models137,138. In order to continue with the characterization of the transfected cells, the expression of NSE was analyzed during the differentiation of cells PC12 and PC12-C1 1. Although this protein is a marker of differentiation, its expression was found in the PC12 and PC12-C1 1 cells undifferentiated. However, the expression of this protein clearly increases in the transfected cells (Figure 16A and 16B). The expression of NSE increases during differentiation in both non-transfected cells and transfected cells. When comparing the expression levels of NSE, it was observed that in PC12-C1 1 cells its expression is increased during the whole differentiation process compared to non-transfected PC12 cells (Figure 16B). The results suggest that the stimulation of the differentiation of transfected cells is due, in part, to the increased expression of NSE.

Example 10. Subcellular localization of Dp71A78 7g in undifferentiated and differentiated cells: colocalization with β-dystroglycan and Grb2.

The subcellular localization of the Dp71 isoforms is very well established in the cells pci259'84'100'102'119'139 and in other cell lines139'140. To determine the function of exons 78 and 79 at the location of Dp71, the subcellular distribution of Dp71A78.79 was analyzed in PC12-C1 cells by indirect immunofluorescence and confocal microscopy. In these analyzes it was found that the distribution of Dp71A78_79 is different to the reported endogenous Dp71 isoforms. This isoform is located almost exclusively in the cellular periphery (Figure 17A and 17G), probably in the plasma membrane, of undifferentiated cells. In the cells differentiated at 6 days with NGF, Dp71A78_79 showed the same localization in the cellular periphery and through the length of the neurites (figure 17D and 17J). Because the location of Dp71A78 79 was determined in stably transfected cells, we decided to also analyze the distribution of Dp71A78 7g in transient transfectants of PC12 cells. The figure 18A shows that the subcellular localization of Dp71A78 79 in transiently transfected cells is the same as that observed in stable transfectants.

On the other hand, it has been reported that the Dp71 isoforms form a complex with β-dystroglycan in PC1284'100'102 and ß-dystroglycan cells with Grb268'69'67. In order to determine if the? 71? 78 79 forms a complex with? -distroglycan, colocalization assays were performed between these two proteins. In the present invention and as previously demonstrated59, β-dystroglycan is located mainly in the cytoplasm of undifferentiated and differentiated PC12-C1 1 cells (Figure 17B and 17E). When determining the percentage of colocalization between Dp71A78 79 and ß-dystroglycan, a value of approximately 10% was obtained both in undifferentiated PC12-C1 cells as well as in differentiated cells, suggesting that there is no significant interaction between these two proteins. Interestingly, a very clear localization of β-dystroglycan is observed in the nuclear periphery, probably in the nuclear membrane of undifferentiated PC12-C1 1 cells (Figure 17B) and the staining pattern changes in the differentiated cells for 6 days (figure 17E). In the case of the Grb2 protein, it was found to be located in the cytoplasm of the PC12-C1 1 undifferentiated and differentiated cells (figure 17H and 17K) and the percentage of colocalization was around 10%, also suggesting that there is no interaction between the? 71? 78 79 and Grb2.

Example 11. Comparison of the subcellular localization of the recombinant proteins Xpress-Dp71A78 79 and Dp71A78 79-V5 in PC12 cells.

As previously described, Dp71 has an actin binding domain in the NH2-terminal region. To rule out the possibility that the epitope "Xpress" added in the NH2-ternal region of Dp71A78.79 was modifying the subcellular location of the Dp71A78 79, the vector pcDNA3.1 / V5-His-TOPO-Dp71A78 79 was constructed, which adds the epitope "V5" in the COOH-terminal region of Dp71A78.79 (Dp71A78 79-V5), as described above, with the object of leaving free the NH2-terminal part of the Dp71A78 7g. Using vectors pcDNA4 / HisMax-TOPO-Dp71A and pcDNA3.1A / 5-His-TOPO- Dp71A78_79 Transient transfection assays were performed on PC 12 cells and analyzed by indirect immunofluorescence and confocal microscopy. In PC12 cells transiently transfected with the pcDNA4 / HisMax-TOPO-Dp71A78.79 vector, the Xpress-Dp71A78_79 protein is located in the cell periphery (Figure 18A) in a manner similar to the location observed in the stable transfectants PC12-C11 . When analyzing the location of the recombinant protein Dp71A78 79-V5, it was found that the distribution of the Dp71A78 79 with the flag in the COOH-terminal is equal to the protein with the "Xpress" flag in the NH2-terminal (figure 18D) . The results obtained indicate that both the flag and its position do not alter the location of the Dp71A78_79.

Example 12. Subcellular localization of Dp71A78 79 in MDCK cells.

Dp71A78 79 is located in the cellular periphery in both the transient transfectants as well as in the stable transfectants of the PC12 cells. In order to determine that this distribution does not depend on the PC12 cell line, transient transfection assays were performed on the MDKC cell line with the vector encoding the recombinant protein Xpress-Dp71A78.79. MDCK cells are immortalized cells that come from the kidney of a dog, a model of origin different from that of PC12 cells. Indirect immunofluorescence assays were performed on the transfected cells and the location of the Dp71A78.79 protein was determined by confocal microscopy. The results obtained are shown in Figure 19A where it can be seen that Dp71A78 79 is found in the cellular periphery of MDCK cells; however, an increase in fluorescence is observed in regions where there is a contact between two transfected MDCK cells (Figure 19D). The results of this part of the work show that Dp71A78 79 is located in the cellular periphery and that this distribution does not depend on the cell line.

The importance of Dp71 isoforms in cell lines, in animal models and in human has been described in several studies48 55'59'85'90 92'93 97'98-111'140'141; however, the biological function of the Dp71 domains was not yet known. In order to study the function of the dp71 domains and considering the composition of the three dystrophin groups (figure 2), a Dp71 mutant was generated by in vitro elimination of the exons 78 and 79, which was named Dp71A78_79. This mutant isoform contains exons 1 and 63 to 70 and 72 to 77 of Dp71 and lacks exons 78 and 79, in addition to exon 71 (figure 2). The absence of exons 78 and 79 does not alter the interaction sites for the DAPs reported to date129,130, however it can modify the characteristics of the protein such as its location, post-translational modifications and / or its folding, so that the expression, function and localization of this protein may be different from that of the Dp71 isoforms reported. In the present invention, for the first time, the elucidation of the biological function of the dp71 domains is contributed.

For this, the vector pcDNA4 / HisMax-TOPO-Dp71A78 7g was constructed (Figure 5A) and stable transfectants of the PC12 cells were generated with this vector. When determining the percentage of differentiation and the average length of neurites, it was found that the stable expression of Dp71A78 79 stimulates the process of neural differentiation of PC12-C1 cells 1 (figure 11). The projection of small neurites in PC12-C11 cells cultured with differentiation medium without NGF was also observed (FIG. 12), demonstrating that the mutant protein Dp71A78 79 of the present invention stimulates differentiation, at least in part, by a pathway of independent differentiation to the NGF; however, NGF is required to stimulate the differentiation of transfected cells. The stable expression of? 71? 78 79 stimulates the process of neural differentiation of PC12 cells in response to NGF, so that exons 1 and 63 to 70 and 72 to 77 of Dp71 are sufficient to induce stimulation of the differentiation of PC12-C1 cells 1 and / or that exons 78 and 79 are not necessary for this function or that these exons have a regulatory function on the isoforms of Dp71a and Dp71ab. On the other hand, the stimulation of the differentiation of PC12-C11 cells may be due to the reduction in the expression of Dp71a in PC12-C11 cells (Figure 13A and 13B). Our results show that the differentiation of PC12 cells does not require the Dp71a isoform or that it has an inhibitory activity for this process. Dp71 e is also decreased in PC12-C1 1 cells, however, its expression increases during differentiation (Figure 13A and 13C).

Previously it was reported that the Dp71 isoforms are necessary for the differentiation of PC12111 cells, in this regard and for the first time it is demonstrated that the stable expression of Dp71A78.79 and the increase in the expression of Dp71 ab / Up71 (figure 13A and 13D) compensate for the function of Dp71a as it occurs with Up400 in models where Dp427142 is absent or that Dp71 ab is the isoform that participates in the process of neuronal differentiation and when its expression is increased in cells PC12-C1 1 allows this cells to accelerate their differentiation. In our model, Dp71 leaves the DAPs complex proteins free allowing the interaction of these proteins with Dp71ab or their integration into adhesion and / or signaling complexes as described below. The delocalisation or relocation of Dp71A78.79 with respect to the endogenous Dp71 isoforms involved in the stimulation of the differentiation of PC12-C1 cells 1. This recombinant protein forms complexes indirectly with some members of the DAPs complex or signaling proteins such as β-dystroglycan, Grb2 and nNOS, stimulating the activation of the NGF pathway and consequently the differentiation of PC12-C1 cells 1.

The increase in the levels of β-dystroglycan expression in the transfected cells (Figure 15A and 15B) may be another reason why PC12-C1 1 cells increase their differentiation. Recently, the importance of β-dystroglycan in cell adhesion, in signal transduction and in the regulation of the actin cytoskeleton was described143. The overexpression of this protein in PC12-C1 cells 1 can contribute to the increase of the adhesion of these cells as well as to the rearrangement of the cytoskeleton proteins, stimulating the differentiation of the transfected PC12 cells. Also, the location of β-dystroglycan in the nuclear periphery of PC12-C1 1 cells (Figure 17B) participates in the activation of differentiation, since this protein forms a complex with Dp71a and other members of the DAPs complex described in nucleus of PC12 cells as well as nuclear matrix proteins119. The decrease in β-dystroglycan staining in the perinuclear region of the PC12 cells differentiated at 6 days (Figure 17E) is due to the decrease in the expression of these proteins in the PC12-C1 1 cells differentiated at 6 days (Figure 15A and 15B), so that the expression and subcellular localization of β-dystroglycan is modulated during the process of neuronal differentiation. On the other hand, the increase in the expression of Dp71 ab also participates in the stimulation of the differentiation of cells PC12-C1 1. Our group previously reported that Dp71ab increases its expression during the differentiation of PC1259 cells, which is also part of the adhesion complex in these cells84. The overexpression of both proteins, β-dystroglycan and Dp71 ab, may be contributing to the efficient differentiation of transfected cells, however the data obtained with the Dp71A78 79 protein produced new findings and knowledge that explain the process of neural differentiation and This is a strategy to stimulate this differentiation in vitro and / or in vivo systems.

In addition to dysregulation in the expression levels of Dp71 a, Dp71e, Dp71ab / Up71 and β-dystroglycan, an increase in the expression of neuron-specific enolase (NSE) was observed in PC12-C1 1 cells (Figure 16) , which has been previously described as a marker protein for neuronal differentiation134,135,136,137'138. Enolase enzymes are involved in the pathway of glycolysis and there are different isoforms (a-, β-, β -enolasa) that have slight variations in their structure and distribution in tissues133,144. The NSE (? -enolasa) is expressed mainly in mature neurons; its expression increases during the development of the brain and during the differentiation and functional maturation of neuronal cells133.

In the present invention, the NSE was analyzed as a marker of neuronal maturation in the differentiation of the PC12-C1 cells 1. We found an increase in its expression in the undifferentiated PC12-C1 cells as well as in the differentiated ones (figure 16). These results demonstrate that PC12-C1 cells are pre-conditioned to differentiate and when NGF is added, these cells respond rapidly initiating the process of neuronal differentiation. Although the level of expression of NSE in untransfected PC12 cells increases during differentiation, it is lower at all times analyzed compared to PC12-C1 1 cells (Figure 16), which partly explains the difference in the index of differentiation and the length of neurites between both cell types.

The localization in the plasma membrane of the recombinant protein Xpress-Dp71A78 79 in cells PC12-C1 1 and DCK demonstrates that exons 78 and 79 participate in the regulation of nuclear localization, cytoplasm Dp71 a, while the amino acid sequence generated by the alternative processing of exon 78 (figure 2) controls the localization of Dp71ab in the cytoplasm and cell periphery as previously described59,120,111, 140 '. In this context, the absence of exons 78 and 79 in the sequence of ?? 71? 78.79, eliminates the phosphorylation site present in Dp71 a that determines its nuclear and cytoplasmic localization, whereas in Dp71 ab the sequence of the 31 amino acids of this isoform that participates in its cytoplasmic localization and cellular periphery. In this way, the membrane location of the Dp71A78. 79 in the present invention may be due, on the one hand, to the absence of the phosphorylation of the threonine residue 3685 present in the Dp71a and on the other hand, to the absence of the 31 amino acids present in the Dp71 ab.

The location of Dp71A78 79 in the plasma membrane is independent of the cell model PC12 or MDCK (see figures 17, 18 and 19); likewise, the location of this protein does not depend on the "Xpress" or "V5" epitope or the NH2- or COOH-terminal position (Figure 18). However, in the MDCK cells the concentration of the Xpress-Dp71A78 7g recombinant protein is observed in the contact regions between the transfected cells (Figure 19D).

In summary, the results described in the present invention demonstrate that the stable expression of Dp71A78 79, stimulates the process of neuronal differentiation of untreated and treated NGF-treated PC12-C1 1 cells, by means of several factors such as the reduction in the expression of Dp71 to Dp71e, the increase in the levels of expression of Dp71ab / Up71, ß-dystroglycan, enolase NSE, the overexpression of Dp71A78 79 during the differentiation of PC12-C1 cells and the over- Activation of the signaling cascade induced by NGF.

On the other hand, our results strongly demonstrate that the protein Dp71A78_79 of the present invention has an effect on the process of neuronal differentiation, providing the molecular basis for the development of a new method with therapeutic potential.

Also, the use of the protein? 71? 78 79 of the present invention turns out to be useful for stimulating neuronal differentiation in cell lines, stem cells in general and neural stem cells in particular, in animal models or in individuals, particularly lines Cells such as PC12 and in humans that present damage to the functioning of the nervous system and / or a certain degree of mental retardation caused, among other causes, by trauma or diseases where there is damage or neuronal degeneration such as Muscular Dystrophy of Duchenne (DMD) ).

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Claims (18)

Claims

1. An isolated nucleotide sequence characterized in that it comprises the sequence SEQ. ID. No.1 or a sequence homologous to it, which codes for the D671A78_7g Dystrophin protein where said sequence lacks exons 78 and 79 but preserves exons 1 and 63 to 77 of the Dp71 dystrophin gene.

2. A mutant protein of Dystrophin characterized by being expressed by the nucleotide sequence of claim 1.

3. A molecular vector characterized by comprising the nucleotide sequence according to claim 1.

4. The molecular vector according to claim 3 which can be referred to as pcDNA4 / HisMax-TOPO-Dp71A78.79 and pcDNA3.1 / V5-His-TOPO-Dp71A78.79 and characterized according to the constituent elements according to figure 5A and 5B .

5. A cell comprising the molecular vector of claim 2.

6. The cell according to claim 5 characterized in that it is a stable transfectant cell that expresses the D671A78_7g Dystrophin protein.

7. A cell according to claims 3 and 4 and which may be a cell of pheochromocytoma origin, such as a PC12 cell.

8. The cell according to claims 6 and 7 characterized by differentiating to a neural line where the expression of the Dp71 ab / Up71 and β-dystroglycan genes is increased, the expression of the Dp71a and Dp71 e genes is decreased and the signaling pathway of NGF is overactivated, determined by the phosphorylation of Erk 1/2.

9. A composition comprising a nucleotide sequence characterized by comprising the sequence SEQ. ID. No.1 or a sequence homologous to it, which codes for the D671A78 7g Dystrophin protein where said sequence lacks exons 78 and 79 but preserves exons 1 and 63 to 77 of the Dp71 dystrophin gene.

10. A composition comprising a molecular vector according to claims 3 and 4.

The composition according to claims 9 and 10 wherein it is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

12. A pharmaceutical composition according to claims 9-1 1 useful for stimulating neural differentiation in cell lines or in subjects.

13. The pharmaceutical composition according to claims 9-12 is useful for stimulating neural differentiation in cell lines or in subjects particularly cell lines such as PC12 and in human subjects that present a certain degree of mental retardation produced by diseases where there is a neuronal degeneration as for example Muscular Dystrophy of Duchenne (DMD).

14. A method for stimulating in vitro neural differentiation which comprises the step of contacting a cell or a group of cells with a composition comprising a nucleotide sequence characterized by comprising the sequence SEQ. ID. No.1 or a sequence homologous to it, which codes for the protein Dp71A7879 Dystrophin where said sequence lacks exons 78 and 79 but retains exons 1 and 63 to 77 of the Dp71 dystrophin gene.

15. The method of claim 14 wherein the cell belongs to the PC12 type.

16. The use of a nucleotide sequence comprising the sequence SEQ. ID. No.1 or a sequence homologous to it, which codes for the D671A78.79 Dystrophin protein where said sequence lacks exons 78 and 79 but preserves exons 1 and 63 to 77 of the Dp71 dystrophin gene to prepare a drug to stimulate Neural differentiation in subjects that present a certain degree of mental retardation produced by diseases where there is a neuronal degeneration.

17. The use according to claim 16 wherein the subjects have a mental retardation produced by Duchenne Muscular Dystrophy (DMD).

18. An experimental study model or system characterized by comprising stable transfectant cells expressing the nucleotide sequence characterized by comprising the sequence SEQ. ID. No.1 or a sequence homologous to it, which codes for the protein Dp71A7879 Dystrophin where said sequence lacks exons 78 and 79 but retains exons 1 and 63 to 77 of the Dp71 dystrophin gene.