MXPA06012693A - Canine ghrh gene, polypeptides and methdos of use. - Google Patents

Abstract

The present invention relates to a canine-pre-proGHRH polypeptide, a canine mature GHRH peptide, an isolated polynucleotide which encodes the canine pre-proGHRH or the canine mature GHRH. The invention also encompasses vectors encoding and expressing pre-proGHRH or GHRH which can be used to treat disease and growth hormone deficiencies by gene therapy in vertebrates, in particular in dogs.

Description

CANINE GHRH GENE, POLYPEPTIDES AND METHODS OF USE FIELD OF THE INVENTION The present invention relates to a canine pre-proGHRH polypeptide, a mature canine GHRH peptide, an isolated polynucleotide that encodes canine pre-proGHRH or mature canine GHRH. In one embodiment, the invention relates to a vector encoding and expressing pre-proGHRH or GHRH. Such a vector can be used to treat diseases and deficiencies of growth hormone by genetic therapy in vertebrates, in particular in dogs. In another embodiment, the invention relates to a method for delivering the GHRH peptide to a vertebrate, in particular to a dog, which comprises injecting the vector expressing in vivo the canine pre-proGHRH or the canine GHRH to the animal host. The invention also relates to a method for the treatment of anemia, cachexia, wound healing, bone healing, osteoporosis, obesity in vertebrates, in particular in dogs. BACKGROUND OF THE INVENTION Growth hormone releasing hormone (GHRH) is a hypothalamic peptide that plays a critical role in controlling the synthesis and secretion of growth hormone (GH) by the anterior pituitary. The GHRH human is a C-terminal amidated peptide of 44 amino acids. In contrast, rat GHRH is a non-amidated peptide, 43 amino acids long, which is only 67% homologous to human GHRH. It is established that GHRH originates from a precursor (pre-proGHRH) that is processed to mature GHRH by removal of signal peptide and proteolytic cleavage in the C-terminal region. The amino acid sequence of the precursor comprises 107 or 108 residues depending on the differential use of two possible splice acceptor sites. The human and rat GHRH gene includes five small exons separated by 4 introns and spans above 10 kilobases in genomic DNA but differs by the position and size of the introns. Patent application EP1052286 illustrates a non-specific method for cloning the canine GHRH gene from a genomic library but the description does not disclose the nucleotide sequence of the gene or does not provide some guidance on how to remove the introns and how to assemble the exon sequences for get the complete coding sequence. In farm animals, GHRH is galoctopoietic without alteration in the composition of the milk, increases the conversion of food to milk and sustains growth, mainly through increased lean body mass. By stimulating the secretion of GH, GHRH increases immune functions in animals. GHRH has a great therapeutic utility in the treatment of cachexia (Bartlett et al. Cancer 1994, 73, 1499-1504 and Surgery 1995, 117, 260-267) in chronic diseases such as cancer, due to abnormalities in the production of growth hormones, in the healing of wounds, bone healing, retardation of the aging process, osteoporosis and in anemia (Sohmiya et al J. Endocrinol, Invest 2000, 23, 31-36 and Clin Endocrinl, 2001, 55, 749-754). Studies have shown that relatively small amounts of GRHG are required to stimulate the production and secretion of GH. However, the use of a heterologous GHRH, which comes from a different animal species, can lead to a less potent stimulation of GH release. The therapeutic administration of the heterologous GHRH peptide can also induce antibody response in the host. The DNA encoding canine GHRH was unknown until the present invention and there was a need to make canine GHRH available in sufficient quantity to treat dogs or other animal species. The citation or identification of any document in this application is not an admission that such a document is available as the prior art for the present invention.

BRIEF DESCRIPTION OF THE INVENTION The present invention is based, in part, in the identification of the applicants of the novel canine pre-proGHRH and the mature GHRH polynucleotide and canine polypeptide sequences. The present invention relates to a canine pre-proGHRH polypeptide, a mature canine GHRH peptide and isolated polynucleotides that encode canine pre-proGHRH or mature canine GHRH. In an advantageous embodiment, the canine pre-proGHRH polypeptide consists essentially of the amino acid residues of SEQ ID NO: 1. In another advantageous embodiment, the mature canine GHRH peptide consists essentially of the amino acid residues of SEQ ID NO: 2. The invention also comprises an isolated polynucleotide, or an antisense strand which is completely complementary thereto, consisting essentially of canine proGHRH, having the sequence of SEQ ID NO: 3 and an isolated polynucleotide, or an antisense strand that is completely complementary thereto, consisting essentially of mature canine GHRH, having the sequence of SEQ ID NO: 4. The polynucleotides can be DNA or RNA molecules. In one embodiment, the invention relates to a vector encoding and expressing the canine pre-proGHRH or the Canine GHRH. Such a vector can be used to treat diseases and deficiencies of growth hormone by genetic therapy in vertebrates. In an advantageous embodiment, the expression vector comprises a polynucleotide encoding a canine pre-proGHRH, wherein the polynucleotide comprises the nucleotide base sequence of SEQ ID NO: 3. In another advantageous embodiment, the expression vector comprises the polynucleotide encoding a mature canine GHRH, wherein the polynucleotide comprises the nucleotide base sequence of SEQ ID NO: 4. In an advantageous embodiment, the nucleotide base sequence is operably linked to a promoter and optionally to an enhancer. In a particularly advantageous embodiment, the invention relates to a vector encoding and expressing canine proGHRH, canine proGHRH deleted from the N-terminal propeptide (corresponding to 31-106 A?) Or canine GHRH wherein a signal sequence of peptide is fused to canine proGHRH, proGHRH-canine suppressed from the N-terminal propeptide (corresponding to 31-106 AA) or canine GHRH. Advantageously, the peptide signal sequence is an insulin-like growth factor (IGF) peptide signal sequence. In a particularly advantageous embodiment, the peptide signal sequence is an IGF-1 peptide signal sequence, more advantageously, a sequence of equine IGF-1 peptide signal. In another advantageous embodiment, the invention comprises a vector encoding and expressing a GHRH containing a peptide signal sequence, advantageously an IGF-1 peptide signal sequence and a proGHRH deleted from the N-terminal propeptide and optionally, the addition of a glycine in the C-terminal. In still another advantageous embodiment, the invention provides a vector backbone containing a peptide signal sequence, advantageously an IGF-1 signal sequence fused to a mature GHRH and a serum albumin optionally linked through a tetrapeptide linker. GHRH can be derived from pigs or cattle as well as dogs. Any of the above vectors can be used to treat diseases and deficiencies of growth hormone by genetic therapy in vertebrates. In advantageous embodiment, the expression vector comprises a polynucleotide encoding a canine pre-proGHRH, wherein the polynucleotide comprises the nucleotide base sequence of SEQ ID NO: 3. In another advantageous embodiment, the expression vector comprises the polynucleotide encoding a mature canine GHRH, wherein the polynucleotide comprises the nucleotide base sequence of SEQ ID NO: 4. In an advantageous embodiment, the nucleotide base sequence is operably linked to a promoter and optionally to an enhancer. In another embodiment, the invention relates to a method for delivering the GHRH peptide to a vertebrate, in particular a dog, comprising injecting the vector that expresses in vivo the canine pre-proGHRH or the canine -GHRH to the animal host. In an advantageous embodiment, the animal host is a dog. In an advantageous embodiment, a formulation comprising a vector that expresses canine GHRH or a canine pre-proGHRH and a pharmaceutically or veterinarily acceptable carrier or vehicle or excipient that facilitates the delivery and expression of canine GHRH or a canine pre-proGHRH in a cell or improves the stability of the vector. Advantageously, the supply is in vivo to an animal, advantageously a vertebrate, more advantageously, a dog. In a more advantageous embodiment, the vector in the formulation comprises the canine pre-proGHRH sequence consisting essentially of SEQ ID NO: 3 or the mature canine GHRH sequence consisting essentially of SEQ ID NO: 4. The invention also provides methods for delivering GHRH to an animal advantageously a vertebrate, more advantageously a dog, comprising injecting a GHRH formulation into the animal. In an advantageous embodiment, the formulation comprises a vector that expresses canine GHRH and a pharmaceutically or veterinarily acceptable excipient carrier or carrier.

The invention also relates to a method for the treatment of anemia, cachexia, wound healing, bone healing, osteoporosis and obesity in vertebrates.

The invention also relates to a method for stimulating the immune response of a vertebrate. It is noted that in this description and particularly in the claims and / or paragraphs, the terms such as "comprises", "understood", "comprising" and the like have the meaning attributed thereto in the US patent law; for example, they can mean "includes", "included", "including", and the like; and that the terms such as consisting essentially of "and" consists essentially of "has the meaning ascribed to these in the American patent law, for example, they allow elements not explicitly cited, but exclude elements that are in the prior art or which affect a basic or novel feature of the invention As used herein, "consisting essentially of" has the meaning to explicitly exclude non-canine GHRH sequences These and other modalities are disclosed or are obvious from, and comprised by, the following Detailed Description BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description, given by way of example, but not proposed to limit the invention only to the specific embodiments described, can be better understood in conjunction with the accompanying drawings, in which: FIG. 1 represents the plasmid map and the encoded ORF of pNB179. The nucleotide sequence of the encoded ORF is SEQ ID NO: 49 and the amino acid sequence of the encoded ORF is SEQ ID NO: 50. FIG. 2 represents the plasmid map and the encoded ORF of pNB209. The nucleotide sequence of the encoded ORF is SEQ ID NO: 26 and the amino acid sequence of the encoded ORF is SEQ ID NO: 27. FIG. 3 represents the plasmid map and the encoded ORF of pNB210. The nucleotide sequence of the ORF encoded in SEQ ID NO: 28 and the amino acid sequence of the ORF encoded in SEQ ID NO: 29. FIG. 4 represents the plasmid map and the encoded ORF of pNB211. The nucleotide sequence of the encoded ORF is SEQ ID NO: 30 is the amino acid sequence of the encoded ORF is SEQ ID NO: 31. FIG. 5 represents the plasmid map and the encoded ORF of pNB212. The nucleotide sequence of the encoded ORF is SEQ ID NO. 32 and the amino acid sequence of the encoded ORF is SEQ ID NO: 33.

FIG. 6 represents the plasmid map and the encoded ORF of pNB213. The nucleotide sequence of the encoded ORF is SEQ ID NO: 34 and the amino acid sequence of the encoded ORF is SEQ ID NO: 35. FIG. 7 represents the plasmid map and the ORF encoded by pNB214. The nucleotide sequence of the encoded ORF is SEQ ID NO: 36 and the amino acid sequence of the encoded ORF is SEQ ID NO: 37. FIG. 8 represents the plasmid map and the encoded ORF of pNB215. The nucleotide sequence of the encoded ORF is SEQ ID NO: 38 and the amino acid sequence of the encoded ORF is SEQ ID NO: 39. FIG. 9 represents the plasmid map and the encoded ORF of pNB216. The nucleotide sequence of the encoded ORF is SEQ ID NO: 40 and the amino acid sequence of the encoded ORF is SEQ ID NO: 27. FIG. 10 represents the plasmid map and the encoded ORF of pNB217. The nucleotide sequence of the encoded ORF is SEQ ID NO: 41 and the amino acid sequence of the encoded ORF is SEQ ID NO: 31. FIG. 11 represents the plasmid map and the encoded ORF of pNB218. The nucleotide sequence of the encoded ORF is SEQ ID NO: 42 and the amino acid sequence of the encoded ORF is SEQ ID NO: 35. FIG. 12 represents the plasmid map and the ORF encoded by pNB219. The nucleotide sequence of the ORF encoded is SEQ ID NO: 43 and the amino acid sequence of the encoded ORF is SEQ ID NO: 37. FIG. 13 represents the plasmid map and the encoded ORF of pNB220. The nucleotide sequence of the encoded ORF is SEQ ID NO: 44 and the encoded ORF amino acid sequence is SEQ ID NO: 39. FIG 13 depicts the plasmid map and the encoded ORF of pNB220. FIG. 14 represents the plasmid map and the encoded ORF of pNB240. The nucleotide sequence of the encoded ORF is SEQ ID NO: 55 and the amino acid sequence of the encoded ORF is SEQ ID NO: 56. FIG. 15 represents the plasmid map and the encoded ORF of pNB239. The nucleotide sequence of the encoded ORF is SEQ ID NO: 57 and the amino acid sequence of the encoded ORF is SEQ ID NO: 58. FIG. 16 represents the plasmid map and the encoded ORF of pNB244. The nucleotide sequence of the encoded ORF is SEQ ID NO: 59 and the amino acid sequence of the encoded ORF is SEQ ID NO: 60. FIG. 17 represents the plasmid map and the encoded ORF of pNB232. The nucleotide sequence of the encoded ORF is SEQ ID NO: 61 and the amino acid sequence of the encoded ORF is SEQ ID NO: 62. FIG. 18 represents the plasmid map and the ORF encoded by pNB245. The nucleotide sequence of the encoded ORF is SEQ ID NO: 63 and the amino acid sequence of the encoded ORE is SEQ ID NO: 64. FIG. 19 represents the plasmid map and the encoded ORF of pNB228. The nucleotide sequence of the encoded ORF is SEQ ID NO: 69 and the amino acid sequence of SEQ ID NO: 70. FIG. 20 represents the plasmid map and the encoded ORF of pNB297. The nucleotide sequence of the encoded ORF is SEQ ID NO: 71 and the amino acid sequence of the encoded ORF is SEQ ID NO: 72. FIG. 21 represents the plasmid map and the encoded ORF of pNB298. The nucleotide sequence of the encoded ORF is SEQ ID NO: 73 and the amino acid sequence of the encoded ORF is SEQ ID NO: 74. FIG. 22 represents the plasmid map and the encoded ORF of pNB299. The nucleotide sequence of the encoded ORF is SEQ ID NO: 76 and the amino acid sequence of the encoded ORF is SEQ ID NO: 77. FIG. 23 represents the plasmid map and the encoded ORF of pNB300. The nucleotide sequence of the encoded ORF is SEQ ID NO: 80 and the amino acid sequence of the encoded ORF is SEQ ID NO: 81. DETAILED DESCRIPTION The present invention is based, in part, on the identification of the applicants of the novel canine pre-proGHRH and canine mature GHRH polynucleotide and polypeptide sequences. Prior to the present invention, there were available GHRH peptides and coding sequences from different animal species such as mouse, rat, pig and bovine. The use of a heterologous GHRH in the dog can lead to a less potent stimulation of GH release and can induce antibody response in the host after repeated injections. DNA encoding canine GHRH was unknown until the present invention and there was a need to make canine GHRH available in sufficient quantity to treat dogs. The present invention relates to a canine pre-proGHRH polypeptide having the following amino acid sequence: H- MPLWFFLVILTLSSGSHSSPPSLPIRIPRYADAIFTNSYRKVLGQLSARKLLQDIMSRQQ GERNREQGAKVRLGRQVDSLWASQKQMALENILASLLQKRRNSQG-OH (SEQ ID NO: 1). The peptide signal sequence (pre-peptide) ranges from Met (l) to Ser (20). Segmentation of the signal peptide can also occur after Ser (19). The number in brackets means the amino acid position in the pre-proGHRH sequence. The H-M and G-OH means that the amino acid Met at the N-terminus and the amino acid Gly at the carboxy terminus of the propeptide are not modified. After cleavage of the preGHRH peptide, the proGHRH peptide is cleaved after the Arg (30) and after Leu (74) to drive the mature GHRH peptide. A variant of the canine pre-proGHRH polypeptide has a substitution of the amino acid Pro at position 2 by the amino acid Leu. The present invention also relates to a mature canine GHRH peptide having the following amino acid sequence: Ri-YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGERNREQGAKVRL-R2 (SEQ ID NO: 2) wherein Ri is hydrogen or H-Met and R2 is OH or NH2. H-Met or H-Y at the N-terminus means that methionine or thyroxine are not modified. The L-OH or L-NH2 at the carboxy terminus of the mature peptide means that the amino acid leucine is either unmodified or aminated. In another embodiment, the invention comprises a mature canine GHRH analogue with improved stability. This analogue has at least one amino acid substitution selected from the group consisting of Tyr (1) to His (1), Ala (2) to Val (2), Gly (15) to Ala (15), Met (27). ) to Leu (27) or Ser (28) to Asn (28.). The number in parentheses means the amino acid position in the mature GHRH sequence. These amino acid substitutions can be introduced into the canine pre-proGHRH polypeptide. It is routine experimentation for one skilled in the art to make such targeted amino acid substitutions. For example, but not by limitation, site-directed mutagenesis of mature canine GHRH polynucleotide can be carried out to obtain a mature mutant canine GHRH peptide with one or more of the amino acid substitutions listed above. The gene can also be chemically synthesized using methods known in the art. The terms "protein", "peptide", "polypeptide" and "polypeptide fragment" are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymer may be linear or branched, it may comprise modified amino acids or amino acid analogs, and may be interrupted by chemical portions other than amino acids. The terms also comprise an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, and any other manipulation or modification, such as conjugation with a labeling or bioactive component. The present invention comprises an isolated polynucleotide encoding the canine pre-proGHRH having the sequence and fragments: 5'- ATGCCACTCTGGCTTGTTCTTCCTGGTGATCCTCACCCTCAGCAGTGGCTCCCACTC TTCCCCGCCATCCCTGCCCATCAGAATCCCTCGGTAtGCAGACGCCATCTTCACC- AACAGCTACCGGAAGGTGCTGGGCCAGCTGTCCGCCCGCAAGCTCCTGCAGGAC ATCATGAGCCGGCAGCAGGGAGAGAGAAACCGGGAGCAAGGAGCAAAGGTACG ACTCGGCCGTCAGGTGGACAGTCTGTGGGCAAGCCAAAAGCAGATGGCATTGGA GAAC TCCTGGCATCCCTGrrACAGAAACGCAGGAACTCCCAAGGATGA-3 '(SEQ ID NO: 3).

The invention also provides a variant of the polynucleotide encoding canine pre-proGHRH having a substitution of nucleotides C and A at position 5 and 6 by T and G, respectively. The present invention also comprises an isolated polynucleotide encoding the mature canine GHRH having the sequence: 5'- TATGCAGACGCCATCTTCACCAACAGCTACCGGAAGGTGCTGGGCCAGCTGTCCG CCCGCAAGCTCCTGCAGGACATCATGAGCCGGCAGCAGGGAGAGAGAAACCGGGAGCAAGGAGCAAAGGTACGACTC-3 '(SEQ ID NO: 4).

A "polynucleotide" is a polymeric form of nucleotides of any length, containing deoxyribonucleotides, ribonucleotides, and the like in any combination. The polynucleotides can have three-dimensional structure, and can perform any function, known or unknown. The term "polynucleotide" includes double-, single-stranded and triple-helical molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide comprises both the double-stranded form and each of the two known or predicted complementary forms. to form the double-stranded form of either the DNA, RNA or hybrid molecule. The following are non-imitative examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA isolated from any sequence, RNA isolated from any sequence, nucleic acid probes and primers. A plinucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogues, uracil, other sugars, and linking groups such as fluororibose and thiolate and nucleotide branches. The nucleotide sequence can also be modified after the polymerization such as by conjugation, with a labeling component. Other types of modifications included in these definitions are terminations, substitution of one or more of the naturally occurring nucleotides with an analogue, and introduction of means to bind the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or support solid An "isolated" polynucleotide or polypeptide is one that is substantially free of the materials with which it is associated in its native environment. By substantially free, at least 50% is proposed, advantageously at least 70%, more advantageously at least 80% and even more advantageously at least 90% free of these materials. The invention further comprises a strand complementary to the GHRH polynucleotide. The complementary strand may be polymeric and of any length, and may contain deoxyribonucleotides, ribonucleotides and analogs in any combination. Hybridization reactions can be performed under conditions of different "severity". Conditions that increase the severity of a hybridization reaction are well known. See for example, "Molecular Cloning: A Laboratory Manual", second edition (Sambroo et al., 1989). Examples of relevant conditions include (in order of increased severity): incubation temperatures of 25 ° C, 37 ° C, 50 ° C and 68 ° C; concentrations of the regulatory solution of 10 x SSC, 6 x SSC, 1 x SSC, 0.1 x SSC (where SSC is 0.15 M NaCl and the 15 M citrate buffer) and its equivalent using other regulatory systems; formamide concentrations of 0%, 25%, 50% and 75%; incubation time from 5 minutes to 24 hours; 1, 2 or more washing steps; wash incubation times of 1, 2 or 15 minutes; and washing solutions of 6 x SSC, 1 x SSC, 0.1 x SSC or deionized water. The invention further comprises polynucleotides that encode functionally equivalent variants and derivatives of the canine GHRH polypeptides and functionally equivalent fragments thereof which may increase, decrease or not significantly affect the properties of the polypeptides encoded in this manner. These functionally equivalent variants, derivatives and fragments exhibit the ability to retain canine GHRH activity. For example, changes in a DNA sequence that do not change the encoded amino acid sequence, as well as those that result in conservative substitutions of amino acid residues, one or a few amino acid deletions or additions, and substitutions of amino acid residues by Amino acid analogues are those that will not significantly affect the properties of the encoded polypeptide. Conservative amino acid substitutions are glycine / alanine; valine / isoleucine / leucine; asparagine / glutamine; aspartic acid / glutamic acid; serine / threonine / methionine; lysine / arginine and phenylalanine / tyrosine / tryptophan. In another embodiment, the invention comprises a canine pre-proGHRH polypeptide variant having at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology or identity to SEQ ID NO: l. In another embodiment, the invention comprises a mature canine GHRH polypeptide variant having at least 97%, at least 97.5%, at least 98%, at least 98.5% or at least 99% homology or identity to the SEQ ID NO: 2. In another embodiment, the invention comprises a variant of polynucleotide that encodes canine pre-proGHRH having at least 86.5%, at least 87%, at least 87.5%, at least 88%, so least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5 %, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97% at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% homology or identity to SEQ ID NO; 3. The invention also comprises a polynucleotide encoding a canine pre-proGHRH analogue. In an advantageous embodiment, the canine pre-proGHRH analog has at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, per at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97% , at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% homology or identity to SEQ ID NO: 1. In another embodiment, the invention comprises a variant of the polynucleotide that encodes mature canine GHRH that has 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, so minus 98%, at least 98.5%, at least 99%, at least 99.5% homology or identity to SEQ ID NO: 4. The invention also comprises a polynucleotide encoding a mature canine GHRH analogue. In an advantageous embodiment, the mature canine GHRH analog has 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5% homology or identity to SEQ ID NO: 2. For the purposes of the present invention, identity or sequence homology is determined by comparing the sequences when aligned to maximize overlap and identity while minimizing sequence spaces. In particular, the identity of Sequence can be determined using any of a number of mathematical algorithms. A non-limiting example of a mathematical algorithm used for the comparison of two sequences is the Karlin & amp; Altschul, Proc. Nati Acad. Sci. USA. 1990; 87: 2264-2268, modified as in Karlin & Altschul, Proc. Nati Acad. Sci. USA. 1993; 90: 5873-5877. Another example of a mathematical algorithm used for comparison of sequences is the algorithm of Myers & Millar, CABIOS 1988; 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) that is part of the GCG sequence alignment software package. When the ALIGN program is used to compare the amino acid sequences, a PAM120 weighted residuals table, a space length penalty of 12 and a space penalty of 4 can be used. Still another useful algorithm for identifying regions of similarity and alignment of Local sequence is the FASTA algorithm as described in Pearson & Lipman, Proc. Nati Acad. Sci. USA. 1988; 85: 2444-2448. Advantageous for use in accordance with the present invention is the WU-BLAST software (Washington University BLAST) version 2.0. The executable programs WU-BLAST version 2.0 for several UNIX platforms can be downloaded from ftp://blast.Wustl.edu/blast/executables. This program is based on WU-BLAST version 1.4, which in turn is based on NCBI-BLAST version 1.4 of the public domain (Altschul &Gish, 1996, Local alignment statistics, Doolittle ed., Methods in Enzymology 266: 640-480, Altschul et al., Journal of Molecular Biology 1990; 215: 403-410; Gish &States, 1993; Nature Genetics 3: 266,272; Karlin &Altschul, 1993; Proc. Nalt, Acad. Sci., USA, 90: 5873-5877, all of which are incorporated by reference herein). In general, the comparison of amino acid sequences is performed by aligning an amino acid sequence and a polypeptide of a known structure with the amino acid sequence of the polypeptide of unknown structure. The amino acids in the sequence are then pitched and groups of amino acids that are homologous are grouped together. This method detects the conserved regions of the polypeptides and takes into account amino acid insertions and deletions. The homology between the amino acid sequences can be terminated by using commercially available algorithms (see also the description of previous homology). In addition to those mentioned otherwise herein, mention is also made of the BLAST, BLAST spaced, BLASTN, BLASTP and PSI-BLAST programs, provided by the National Center for Biotechnology Information. These programs are widely used in the art for this purpose and can align homologous regions of two amino acid sequences.

In all on-site search programs, the spaced alignment routines are integral to the same database search. The spacing can be flipped if desired. The error sanction (Q) for a space of length one is Q = 9 for proteins and BLASTP, and Q = 10 for BLASTN, but it can be changed to any integer number. The penalty for error residue 'to extend a space (R) is R = 2 for proteins and BLASTP, R = 10 for BLASTN,. but it can be changed to any whole number. Any combination of values for Q and R can be used in order to align sequences to maximize identity overlap while minimizing sequence spaces. The error amino acid comparison matrix is BLOSUM62, but other amino acid comparison matrices such as PAM can be used. Alternatively or additionally, the term "homology" or "identity", for example, with respect to a nucleotide or amino acid sequence, may indicate a quantitative measure of homology between two sequences. The percent sequence homology can be calculated as (Nref - Ndif) * 100 / Nef, where Ndií is the total number of non-identical residues in the two sequences when they are aligned and where rer is the number of residues in one of the sequences. Accordingly, the AGTCAGTC DNA sequence will have a sequence identity of 75% with the sequence AATCAATC (ref = 8; NdiJf = 2). Alternatively or additionally, the "homology" or "identity" with respect to sequences may refer to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the two sequences wherein the alignment of the two sequences can be terminated according to the algorithm of Wilbur and Lipman (Wilbur &Lipman, Proc Nati Acad Sci USA 1983, 80: 726, incorporated herein by reference), for example, using a window size of 20 nucleotides , a word length of 4 nucleotides, and a space penalty of 4, and computer-aided analysis and interpretation of the sequence data included in the training can be conveniently performed using commercially available programs (e.g., Intelligenetics ™ Suite, Intelligenetics Inc. CA). When the RNA sequences are said to be similar, or have a degree of identity or sequence homology with the DNA sequence, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. Thus, RNA sequences are within the scope of the invention and can be derived from DNA sequences, by thymidine (T) in the DNA sequence that is considered equal to uracil (U) in RNA sequences. And, without undue experimentation, the expert person You can check with many other programs or references to determine the homology percent. The invention further comprises canine GHRH polynucleotides contained in a vector molecule or an expression vector and operably linked to a promoter element and optionally an enhancer. The invention further comprises canine GHRH polynucleotides contained in a vector molecule or an expression vector wherein a peptide signal sequence is fused to canine proGHRH, canine proGHRH deleted from the N-terminal propeptide (corresponding to 31-106 AA ) or the canine GHRH. Advantageously, the peptide signal sequence is an insulin-like growth factor (IGF) peptide signal sequence. In a particularly advantageous embodiment, the peptide signal sequence is an IFG-1 peptide signal sequence, more advantageously, an equine IGF-1 peptide signal sequence. One skilled in the art can optimize the expression of canine proGHRH, the canine proGHRH deleted from the N-terminal propeptide (corresponding to 31-106 AA) or the canine GHRH nucleotide sequence fused to the peptide signal sequence by removing the sites of Cryptic splicing, for example, by adapting codon usage by introducing the Kozak consensual sequence before the start codon to improve expression.

In an advantageous embodiment, the promoter is the promoter of the cytomegalovirus immediate early gene (CMV). In another advantageous embodiment, the promoter and / or enhancer elements are indecipherable by oxygen. Examples of oxygen-inducible promoters and / or enhancers that can be used in the methods of the present invention include, but are not limited to, the early growth-1 (Egrl) see response promoter, for example (see, for example. , Park et al, J Clin Invest, 2002 August; 110 (3): 03-1), inducible augmentators for hypoxia inducible factor (HIF) (see, for example, Cuevas et al., Cancer Res. October 15, 2003).; 63 (20): 6877-84) and the promoters of Mn-superoxide dismutase (Mn-SOD) (see, for example, Gao et al., Gene. 1996 October 17; 176 (1-2): 269-70) . In another embodiment, the enhancers and / or promoters include various cell or tissue-specific promoters (eg, muscle, endothelial cell, liver, somatic cell or stem cell), various viral promoters and boosters and various GHRH DNA sequences. specific for each species of animal. For example, if canine GHRH is expressed in a canine muscle cell, the enhancers and / or promoters can be specific to a canine muscle cell in order to optimize the expression of canine GHRH. Examples of promoters and augmentators Muscle specificities have been described and are known to one skilled in the art (see, for example, Li et al, Gene Ther, December 1999; 6 (12): 2005-11; Li et al., Nat Biotechnol, March 1999).; 17 (3): 241-5 and Loirat et al., Virology, July 20, 1999; 260 (1): 74-83; the description of which are incorporated by reference in their totalities). Promoters and augmentators that can be employed in the present invention include, but are not limited to, LTR or Rous sarcoma virus, HSV-1 TK early or late SV40 promoter, major late adenovirus.

(MLP), phosphoglycerate kinase, metallothionein, α-1-antitrypsin, albumin, collagenase, elastase I, β-actin, β-globin, β-globin, α-fetoprotein, muscle creatine kinase. A "vector" refers to a plasmid or recombinant DNA or RNA virus comprising a heterologous polynucleotide that is delivered to a target cell, either in vitro or in vivo. The heterologous polynucleotide may comprise a sequence of interest for therapy purposes, and may optionally be in the form of an expression cassette. As used herein, a vector needs not to be capable of replication in the final target cell or subject. The term includes cloning vectors and viral vectors are also included.

The term "recombinant" means a semi-synthetic polynucleotide, or synthetic origin that either does not occur in nature or is linked to another polynucleotide in an arrangement not found in nature. "Heterologist" means a derivative of an entity genetically distinct from the rest of the entity to which it is being compared. For example, a polynucleotide can be placed by genetic engineering techniques on a plasmid or vector derived from a different source, and is a heterologous polynucleotide. A promoter removed from its active coding sequence and operably linked to a coding sequence different from the native sequence is a heterologous promoter. The polynucleotides of the invention may comprise additional sequences, such as additional coding sequences within the same transcription unit, control elements such as promoters, ribosome binding sites, polyadenylation sites, additional transcription units under the control thereof. or a different promoter, sequences that allow cloning, expression, homologous recombination and transformation of a host cell and any construction of such a class as may be desirable to provide embodiments of this invention. The present invention comprises a vector that expresses pre-proGHRH, mature GHRH, proGHRH or variants or analogs or fragments. For mature GHRH or proGHRH, the nucleotide sequence encoding the peptide is preferably immediately preceded by a nucleotide sequence in the structure encoding a peptide signal to obtain the GHRH secreted in extracellular media. The signal sequence may be the natural sequence of the pre-proGHRH or a peptide signal of a secreted protein, for example the signal peptide of the tissue plasminogen activating protein (Tpa), in particular the human tPA (S. Friezner Degen et al J. Biol. Chem. 1996, 261, 6972-6985, R. Rickles et al J. Biol. Chem. 1988, 263, 1563-1569, D. Berg., And collaborators Biochem. Biophys. Res. Commun. 1991, 179, 1289-1296), or the insulin-like growth factor 1 (IGF1) signal peptide, in particular equine IGF1 (K. Otte et al., Gen. Comp.Endocrinol., 1996, 102 (1), 11 -15), the canine IGF1 (P. Delafontaine et al., Gene 1993, 130, 305-306), the feline IGF1 (Wo-A-03/022886), the bovine IGF1 (S. Lien et al., Mamm. 2000, 11 (10), 877-882), porcine IGF1 (M. Muller et al., Nucleic Acids Res. 1990, 18 (2), 364), chicken IGF1 (Y. Kajimoto et al., Mol. Endocrinol. 1989, 3 (12), 1907-1913), the I GF1 of turkey (GenBank access number AF074980). The signal peptide of IGF1 can be natural or optimized, in particular optimized by removing cryptic splice sites and / or by adapting codon usage. For the mature GHRH Gly and Arg can be added at the C-terminus of the peptide in the coding polynucleotide sequence in order to obtain the amidation. The elements for the expression of canine GHRH are advantageously present in an inventive vector. Minimally, this comprises, consists essentially of, or consists of an initiation codon (ATG), such as a stop codon and a promoter, and optionally also a polyadenylation sequence for certain vectors such as plasmid and certain viral vectors, example, viral vectors other than poxviruses. When the polynucleotide encodes a polyprotein fragment, for example canine GHRH, advantageously, in the vector, an ATG is placed 5 'in the reading frame and a stop codon is placed in 3'. Other elements to control expression may be present, such as enhancer sequences, stabilizing sequences, such as intron and signal sequences that allow the secretion of the protein. The methods for making and / or administering a vector or recombinants or plasmid for the expression of gene products of the genes of the invention either in vivo or in vivo can be any desired method, for example, a method that is by, or analogous to, the methods disclosed in, or disclosed in the documents cited in: U.S. Patent Nos. 4, 603,112; 4,769,330; 4,394,448; 4,722,848; 4,745,051; 4,769,331; 4,945,050; 5,494,807; 5,514,375; 5,744,140; 5,744,141; 5,756,103; 5,762,938; 5,766,599; 5,990,091; 5,174,993; 5,505,941; 5,338,683 5,494,807; 5,591,639; 5,589,466; 5,677,178; 5,591,439 5,552,143; 5,580,859; 6,130,066; 6,004,777; 6,130,066; 6,497,883; 6,464,984; 6,451,770; 6,391,314; 6,387,376 6,376,473; 6,368,603; 6,348,196; 6,306,400; 6,228,846 6,221, 362; 6.217, 883; 6,207,166; 6,207,165; 6,159,477; 6,153,199; 6,090,393; 6,074, 649; 6,045,803; 6,033,670; 6,485,729; 6,103,526; 6,224,882; 6,312,682; 6,348,450 and 6,312,683; US Patent Application Serial Number 920,197, filed October 16, 1986; WO 90/01543; W091 / 11525; WO 94/16716; WO 96/39491; WO 98/33510; EP 265785; EP 0 370 573; Andreansky et al., Proc. Nati Acad. Sci. USA 1996; 93: 11313-11318; Ballay et al., EMBO J. 1993; 4: 3861-65; Felgner et al., J. Biol. Chem. 1994; 269: 2550-2561; Frolov et al., Proc. Nati Acad. Sci. USA 1996; 93: 11371-11377; Graham, Tibtech 1990; 8: 85-87; Grunhaus et al., Sem. Virol. 1992; 3: 237-52; Ju et al, 'Diabetología 1998; 41: 736-739; Kitson et al., J. Virol. 1991; 65: 3068-3075; McClements et al., Proc. Nati Acad. Sci. USA 1996; 93: 11414- 11420; Moss, Proc. Nati Acad. Sci. USA 1996; 93: 11341-11348; Paoletti, Proc. Nati Acad. Sci. USA 1996; 93: 11349-11353; Pennock et al., Mol. Cell. Biol. 1984; 4: 399-406; Richardson (Ed), Methods in Molecular Biology 1995; 39, "Baculovirus Expression Protocols", Humana Press Inc .; Smith et al., '1983) Mol. Cell. Biol. 1983; 3: 2156-2165; Robertson et al., Proc. Nati Acad. Sci. USA 1996; 93: 11334-11340; Robinson and collaborators, Sem. Im unol. 1997; 9: 271; and Roizman, Proc. Nati Acad. Sci. USA 1996; 93: 11307-11312. Thus, the vector in the invention can be any recombinant virus or virus vector, such as a poxvirus (e.g., vaccinia virus, avipox virus, canaripox virus, fowlpox virus, raconpox virus, swinepox virus, etc.), adenovirus (e.g., human adenovirus, canine adenovirus), herpesvirus (e.g., herpesvirus) canine), baculovirus, retroviruses, etc. (as the documents incorporated herein by reference); or the vector can be a plasmid. As cited herein and incorporated herein by the reference documents, in addition to providing examples of vectors useful in the practice of the invention, can also provide non-canine GHRH peptides or fragments thereof, eg, mature GHRH peptides not canines, non-canine pre-proGHRH peptides, non-canine pre-GHRH peptides, non-canine proGHRH peptides or fragments thereof, cytokines, etc., which are expressed by the vector or vectors in, or included in, the compositions of the invention. The present invention also relates to preparations comprising vectors, such as expression vectors, for example, therapeutic compositions. The preparations may comprise, consist essentially of, or consist of, one or more vectors, for example, expression vectors, such as expression vectors -in vivo, comprising, consisting essentially of, or consisting of (and advantageously express) one or more of the GHRH polynucleotides. Advantageously, the vector contains and expresses a polynucleotide which includes, consists essentially of, or consists of a coding region encoding canine GHRH, in a pharmaceutically or veterinarily acceptable carrier, excipient or carrier. Thus, according to one embodiment of the invention, the other vector or vectors er. the preparation comprises, consists essentially of, or consists of a polynucleotide encoding and under appropriate circumstances the vector expresses one or more other canine GHRH proteins or a fragment thereof. I agree cor. another embodiment, the vector or vectors in the preparation comprises, or consists essentially of, or consists of polynucleotide (s) encoding one or more 'proteins or fragments (s) thereof of the canine GHRH, the vector or vectors have expression of the polynucleotide (s). The Inventive preparation advantageously comprises, consists essentially of, c consists of, at least two vectors comprising, consisting essentially of, or consisting of and advantageously also advantageously expressing in vivo or under appropriate conditions or suitable conditions in a suitable host cell , polynucleotides of different canine GHRH isolates that encode the same proteins and / or for different proteins, but advantageously for the same proteins. Preparations containing one or more vectors containing, consisting essentially of, or consisting of polynucleotides encoding, and advantageously expressing, advantageously in vivo, canine GHRH peptide, fusion proteins or an epitope thereof. The invention is also directed to mixtures of vectors that contain, consist essentially of, or consist of the coding for, and express, different GHRH, eg, GHRH from different species such as, but not limited to, cats, cows, goats, humans, mice, monkeys, pigs, rats and sheep, in addition to dogs. According to one embodiment of the invention, the expression vector is a viral vector, in particular an expression vector in vivo. In an advantageous embodiment, the expression vector is an adenovirus vector. Advantageously, the adenovirus is a human Ad5 vector, an adenovirus El-deleted and / or an E3-deleted. In a particular embodiment, the viral vector is poxvirus, for example, a vaccinia virus or an attenuated vaccinia virus (e.g., MVA, a modified Ankara strain obtained after more than 570 passages of the Ankara vaccine strain in chicken embryo fibroblasts; see Stickl &Hochstein-Mintzel, Munch, Med. Wschr., 1971, 113, 1149-1153; Sutter et al., Proc. Nati, Acad. Sci. USA, 1992, 89, 10847-10851 available as ATCC VR-1508, or NYVAC, see U.S. Patent No. 5,494,807, for example, Examples 1 to 6 and et se of U.S. Patent No. 5,494, 807 which discusses the construction of NYVAC, as well as the variation of NYVAC with additional ORFs deleted from the Copenhagen vaccinia virus genome, as well as the insertion of heterologous coding nucleic acid molecules at sites of this recombinant, and also, the use of matched promoters, see also W096 / 40241), a avipox or ur virus. attenuated avipox virus (e.g., canaripox, fowlpox, dovepox, pigeonpox, quailpox, ALVAC or TROVAC; see, for example, U.S. Patent Nos. 5,505,941, 5,494,807), swinepox, raconpox, camelpox, or myxomatosis virus. According to another embodiment of the invention, the poxvirus vector is a canaripox virus or a fowlpox virus vector, advantageously an attenuated canaripox virus. or fowlpox virus. In this regard, it is made available to the canaripox of the ATCC under accession number VR-111. The attenuated canaripox viruses are described in U.S. Patent No. 5,756,103 (ALVAC) and WO01 / 05934. Numerous strains of fowlpox virus vaccination are also available, for example, the DIFTOSEC CT strain marketed by MERIAL and the NOBILIS VARIÓLE vaccine marketed by INTERVET; and, the reference is also made to U.S. Patent No. 5,766,599 which belongs to the attenuated fowlpox strain TROVAC. For information for the method for generating recombinants thereof and how to administer recombinants thereof, the skilled person can refer to the documents cited herein and to W090 / 12882, for example, as the mention of the vaccinia virus is made from the U.S. Patent Nos. 4,769,330, 4,722,848, 4,603,112, 5,110,587, 5,494,807, and 5,762,938 initially; as to fowlpox, the mention is made of U.S. Patent Nos. 5,174,993, 5,505,941 and US-5,766,599 initially; as to canaripox the mention is made to the North American patent No. 5,756,103 in ter alia; as to swinepox the mention is made of the North American patent No. 5,382,425 inter alia; and, as with raconpox, the reference is made to WO00 / 03030 inter alia. When the expression vector is a vaccinia virus, the insertion site or sites for the polynucleotides or polynucleotides that are expressed are advantageously in the ge thymidine kinase (TK) or the insertion site, the hemagglutinin gene (HA) or insertion site, the region encoding the inclusion body of type A (ATI); see also the documents cited here, especially those that belong to the vaccinia virus. In the case of canaripox, advantageously the insertion sites or sites are ORF (s) C3, C5 and / or C6; see also the documents cited herein, especially those pertaining to the canaripox virus. In the case of fowlpox, advantageously the insertion sites or sites are ORFs F7 and / or F8; see also the documents cited herein, especially those pertaining to the fowlpox virus. The insertion site or sites for the MVA virus area advantageously as in several publications, including Carroíl M. W. et al., Vaccine, 1997, 15 (4), 387-394; Stittelaar K. J. et al., J. Virol., 2000, 74 (9), 4236-4243; Sutter G. et al., 1994, Vaccine, 12 (11), 1032-1040; and, in this respect it is also observed that the complete MVA genome is described in Antoine G., Virology, 1998, 244, 365-396, which allows the skilled person to use other insertion sites or other promoters. Advantageously, the polynucleotide that is expressed is inserted under the concrol of a specific poxvirus promoter, for example, the 7.5 kDa vaccinia promoter.

(Cochran et al., J. Virology, 1985, 54, 30-35), the vaccinia promoter I3L (Riviere et al., J. Virology, 1992, 66, 3424-3434), the vaccinia HA promoter (Shida, Virology , 1986, 150, 451-457), the promoter of cowpox ATI. { Funahashi et al., J. Gen. Virol., 1988, 69, 35-4"71 ', the vaccinia promoter H6 (Taylor J. et al., Vaccine, 1988, 6, 504-508; Guo P. et al. J. Virol., 1989, 63, 4189-4198, Perkus M. et al, J. Virol., 1989, 63, 3829-3836), in a particular embodiment, the viral vector is an adenovirus, such as a human adenovirus (HAV) or a canine adenovirus (CAV) In one embodiment, the viral vector is a human adenovirus, in particular an adenovirus of serotype 5, made incompetent for replication by a deletion in the El region of the viral genome, in particular from about nucleotide 459 to about nucleotide 3510 by reference to the hAd5 sequence disclosed in Genbank under accession number M73260 and in the referenced publication J. Chroboczek et al., Virol. 1992, 186, 280-285.The deleted adenovirus is propagated in 293 that expresses El (F. Graham et al., J. Gen. Virol. 1977, 36, 59-72) or c. PER cells, in particular PER.C6 (F. Falloux et al., Human Gene Therapy 1998, 9, 1909-1917). The human adenovirus can be deleted in the E3 region, in particular from about nucleotide 28592 to about nucleotide 30470. The deletion in region? l can be in combination with a deletion in the _E3 region (see, for example, J. Shriver et al., Nature, 2002, 415, 331-335). , F. Graham et al., Methods in Molecular Biology Vol. 7: Gene Transfer and Expression Protocols Edited by E. Murray, The Human Press Inc, 1991, p 109-128; Y. lian et al., Proc. Nati Acad. Sci 1997, 94, 2587-2592; S6, 133,028; US6,692,956; S Tripathy et al., Proc. Nati, Acad. Sci. 1994, 91 11557-11561; B. Tapnell Adv. Drug Deliv. Rev. 1993, 12 185-199; X. Danthinne et al., Gene Thrapy 2000, 7 1707-1714; K. Berkner Bio Techniques 1988, 6, 616-629; K Berkner et al., Nuci, Acid Res. 1983, 11, 6003-6020 C Chavier et al, J. Virol. 1996, 70, 4805-4810) The insertion sites can be the El and / or E3 sites (region 'ever.tuaimeat after partial or complete deletion of the El and / or E3 regions.) Advantageously, when the expression vector is an adenovirus, the polynucleotide that is expressed is inserted under the control of a functional promoter in eukaryotic cells. icas, such as a strong promoter, preferably an early and immediate cytomegalovirus gene promoter (CMV-IE promoter), in particular the promoter / promoter region from about nucleotide -734 to about nucleotide +7 in M. Boshart et al., Cell 1985, 41, 521-530 or the enhancer / promoter region of the pCl vector of Promega Corp. The CMV-IE promoter is advantageously of murine or human origin. The promoter of the elongation factor can also be used. In a particular embodiment a promoter regulated by hypoxia, for example the HRE promoter described in K. Boast et al., Human Gene Therapy 1999, 13, 2197-2208), may be used. A specific muscle promoter can also be used (X. Li et al, Nat Biotechnol, 1999, 17, 241-245). Strong promoters are also discussed herein in relation to plasmid vectors. In one embodiment, a splice sequence can be located downstream of the enhancer / promoter region. For example, isolated intron 1 of the CMV-IE gene (R. Stenberg et al., J. Virol. 1984, 49, 190), the intron isolated from the rabbit or human beta-globin gene, in particular intron 2 of the b-globin gene, the isolated intron of the immunoglobulin gene, a splicing sequence of the SV40 early gene or the chimeric intron sequence isolated from the pCl vector of Promega Corp. comprising the donor sequence of human ß-globin fused to the sequence mouse immunoglobulin acceptor (from about nucleotide 890 to about 1022 nucleotide in Genbank under accession number CVU47120). A poly (A) sequence and terminator sequence can be inserted downstream of the polynucleotide being expressed, for example a bovine growth hormone gene, in particular, of about nucleotide 2339 from about nucleotide 2550 in Genbank under the accession number BOVGKRH, a rabbit β-globin gene or a polyadenylation signal of the SV40 late gene. In another embodiment, the viral vector is a canine adenovirus, in particular a CAV-2 (see, for example, L. Fischer et al., Vaccine, 2002, 20, 3485-3497, U.S. Patent No. 5,529,780, U.S. Pat. No. 5,688,920; PCT Application No. W095 / 14102). For CAV, the insertion sites may be in the E3 region and / or in the region located between the E4 region and the right ITR region (see, US Pat. No. 6,090,393, US Patent No. 6,156,567). In one embodiment the insert is under the control of a promoter, such as a gene promoter "immediately remodel from cytomegalovirus (CMV-IE promoter) or a promoter already described for a human adenovirus vector." A sequence of poly (A) and The terminator sequence can be inserted downstream of the polynucleotide that is expressed, eg, a bovine growth hormone gene or a polyadenylation signal of rabbit ß-globin gene.In another particular embodiment of the viral vector is a herpesvirus such as a canine herpesvirus (CHV) or a feline herpesvirus (FHV). For CHV, the insertion sites can be in particular in the thymidine kinase gene, in the 0RF3, c in the UL43 ORF (see, U.S. Patent No. 6,159,477). In one embodiment the polynucleotide that is expressed is inserted under the control of a functional promoter in eukaryotic cells, advantageously a CMV-IE promoter (murine or human). In a particular embodiment, a hypoxia-regulated promoter, for example, the HRE promoter described in K. Boast et al., Human Gene Therapy 1999, 13, 2197-2208), may be used. A poly (A) sequence and terminator sequence can be inserted downstream of the polynucleotide being expressed, for example, bovine growth hormone or a polyadenylation signal of the rabbit β-globin gene. According to still further embodiment of the invention, the expression vector is a plasmid vector or a DNA plasmid vector, in particular an expression vector in vivo. In a non-limiting, specific example, the plasmid pVRl020 or 1012 (VICAL Inc., Luke C. et al., Journal of Infectious Diseases, 1997, 175, 91-97; Hartikka J. et al., Human Gene Therapy, 1996, 7 , 1205-1217, see, for example, U.S. Patent Nos. 5,846,946 and 6,451,769) can be used as a vector for the insertion of a polynucleotide sequence. The plasmid pVR1020 is derived from pVR1012 and contains the human tPA signal sequence. In one embodiment the signal tPA hu ana comprises amino acid Mil) l amino acid S (23) in Genbank under accession number HUMTPA14. In another specific non-limiting example, the plasmid used as a vector for the insertion of a polynucleotide sequence may contain the equine IGF1 signal peptide sequence from the amino acid M (24) to the amino acid (48) in Genbank under the access number U28070. Additional information on DNA plasmids that may be consulted or used in practice are found, for example, in US Pat. Nos. 6,852,705; 6,818,628; 6,586,412; 6,576,243; 6,558,674; 6,464,984; 6,451,770; 6,376,473 and 6,221,362. The term "plasmid" covers any unit of DNA transcription comprising a polynucleotide according to the invention and the elements necessary for its expression in vivo in a cell or cells of the desired host or target; and, in this regard, it is noted that a circular, supercoiled or non-coiled supercoiled plasmid, as well as a linear form, are proposed to be within the scope of the invention. Each plasmid comprises or contains or consists essentially of, in addition to the polynucleotide encoding mature canine GHRH, canine pre-proGHRH, canine proGHRH with a heterologous peptide sequence, the propeptide GHRH deleted from N-terminal propeptide (corresponding to 31-106 AA), variant, analog or fragment, operably linked to a promoter or under the control of a promoter or dependent on a promoter. In general, it is advantageous to employ a strong functional promoter in eukaryotic cells. The preferred strong promoter is the immediate early cytomegalovirus (CMV-IE) promoter of human or murine origin, or optionally having another origin such as rat or guinea pig. The CMV-IE promoter can comprise the current promoter part, which may or may not be associated with the augmenting part. Reference can be made to EP-A-260 148, EP-A-323 597, US Patent Nos. 5,168,062, 5,385,839 and 4,968,615, as well as PCT Application No. 087/03905. The CMV-IE promoter is advantageously a human CMV-IE (Boshart M. et al., Cell., 1985, 41, 521-530) or murine CMV-IE. Er. More generally, the promoter has either a viral or cellular origin. A strong viral promoter other than CMV-IE that can be usefully employed in the practice of the invention is the early / late promoter of the SV40 virus or the LTR promoter of the Rous sarcoma virus. A strong cell promoter that can be usefully employed in the practice of the invention is the promoter of a skeletal site gene, such as, for example, the desmin promoter (Kwissa M. et al., Vaccine, 2000, 18, 2337-2344) , or the Actin promoter (Miyazaki J. et al., Gene, 1989, 79, 269-277). Functional subfragments of these promoters, ie, portions of these promoters that maintain appropriate promoter activity, are included within the present invention, eg, CMV-IE promoters truncated according to PCT application No. WO98 / 00166 or US Pat. No. 6,156,567 can be used in the practice of the invention. A promoter in the practice of the invention consequently includes derivatives and subfragments of a full-length promoter that maintains an appropriate promoter activity and therefore function as a promoter, preferably that promotes activity substantially similar to that of the current promoter or promoter. full length from which the derivative or subfragment is derived, for example akin to the activity of the truncated CMV-IE promoters of US Pat. No. 6,156,567 to the activity of the full-length CMV-IE promoters. Thus, a CMV-IE promoter in the practice of the invention may comprise or consist essentially of or consist of the promoter portion, the full length promoter and / or the enhancer portion of the full length promoter, as well as derivatives and subfragments. Preferably, the plasmids comprise or consist essentially of other control elements. expression. It is particularly advantageous to incorporate stabilizing sequence (s) for example, intron sequence (s), preferably the first intron of hCMV-IE (PCT application No. WO89 / 01036), intron II of the β-globin gene of rabbit (van Ooyen et al., Science, 1979, 206, 337-3 4). As for the polyadenylation signal (polyA) for the plasmids and viral vectors other than poxviruses, the use can be made of the poly (A) signal of the bovine growth hormone gene (bGH) (see, to North American patent No. 5, -122, 58), or the poly (A) signal of the rabbit ß-globin gene or the poly (A) signal of the SV40 virus. According to an advantageous embodiment of the invention, the expression vector comprises the polynucleotides encoding the IGF1 signal peptide and the GHRH propeptide deleted from the N-terminal propeptide (corresponding to 31-106 AA). According to another advantageous embodiment of the invention, the expression marker comprises the polynucleotides encoding the signal peptide of IGF1 and the mature GHRH (corresponding to 31-74 AA). In another advantageous embodiment, the invention comprises a vector encoding and expressing a GHRH containing a peptide signal sequence, advantageously an IGF-1 peptide signal sequence and a proGHRH deleted from the N-terminal propeptide and optionally, the addition of a glycine in the C-terminal. In still another advantageous embodiment, the invention provides a vector backbone containing a peptide signal sequence, advantageously a functional IGF-1 signal sequence to a mature GHRH and a serum albumin optionally linked through a tetrapeptide linker. GHRH can be derived from pigs or cattle as well as dogs. According to another embodiment of the invention, the expression vectors are expression vectors used for the expression of an in vitro protein in an appropriate cell system. The expressed proteins may be harvested in, or on the culture pool after, or not after secretion (if there is secretion typically occurs or a cell lysis is performed), optionally concentrated by concentration methods such as ultrafiltration and / or purified by means of purification, such "affinity, ion exchange or gel filtration type chromatography methods." Host cells that can be used in the present invention include, but are not limited to, muscle cells, keratinocytes, myoblasts, Chinese hamster ovary cells (CHO), vero cells, BHK21, sf9 cells and the like It is understood to one skilled in the art that the conditions for culturing a host cell vary according to the particular gene and that the Routine experimentation is sometimes necessary to determine the optimal conditions to cultivate canine GHRH depending on the host cell. For example, the vector encoding canine GHRH can be transformed into myoblasts (which can be obtained from the muscle tissue of the animal in need of treatment) and the transformed myoblasts can be transplanted into the animal. As a result, myoblasts genetically engineered to express recombinant canine GHRH can secrete the hormone in the animal's blood. In another example, keratinocytes can also be transformed with a vector encoding GHRH and transplanted into the animal, resulting in the secretion of canine GHRH into the circulation. A "host cell" denotes a prokaryotic or eukaryotic cell that has been genetically altered, or is capable of being genetically altered by the administration of an exogenous polynucleotide, such as a plasmid or recombinant vector. When referring to genetically altered cells, the term refers to both the originally altered cell and the progeny thereof. Polynucleotides comprising a desired sequence can be inserted into a suitable cloning or expression vector, and the vector in turn can be introduced into a host cell suitable for replication and amplification. The polynucleotides can be introduced into host cells by any means known in the art. Vectors containing polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including direct uptake, endocytosis, transfection, f-coupling, electroporation, transfection using calcium chloride, rubidium chloride, phosphate calcium, DEAE-dextran or other substances; bombing of microprojectiles; lipofection; and infection (where the vector is infectious, for example, a retroviral vector). The choice of introduction of vectors or polynucleotides will often depend on the characteristics of the host cell. In an advantageous embodiment, the invention provides for the administration of a therapeutically effective amount of a formulation for the delivery of the expression of a canine GHRH to a target cell. The determination of the therapeutically effective amount is routine experimentation for one of ordinary skill in the art. In one embodiment, the formulation comprises an expression vector comprising a polynucleotide expressing canine GHRH and a pharmaceutically or veterinarily acceptable carrier, vehicle or excipient. In an advantageous embodiment, the pharmaceutically or veterinarily acceptable carrier, vehicle or excipient facilitates transcription and / or improves the preservation of the vector or protein. The pharmaceutically or veterinarily acceptable carrier or carriers or excipients are well known to one skilled in the art. For example, a pharmaceutically or veterinarily acceptable carrier or vehicle or excipient may be a 0.9"NaCl solution (e.g., saline) or a phosphate buffer solution Another pharmaceutically or veterinarily acceptable carrier or vehicle or excipient that may be used for the methods of this invention include, but are not limited to, poly- (L-glutamate) or polyvinylpyrrolidone The pharmaceutically or veterinarily acceptable carrier or vehicle or excipient can be any compound or combination of compounds that facilitate the administration of the vector (or expressed protein from an inventive vector in vitro), advantageously, the carrier, vehicle or excipient can facilitate transfection and / or enhance the preservation of the vector (or protein). Dosages and dose volumes are discussed herein in the general description and can also be determined by the skilled person from this description read in conjunction with knowledge in the art, without any undue experimentation. Cationic lipids containing a salt of quaternary ammonium which are advantageously, but not exclusively, suitable for plasmids, are advantageously those having the following formula: wherein Rj is a saturated or unsaturated straight chain aliphatic radical having 12 to 18 carbon atoms, R2 is another aliphatic radical containing 2 or 3 carbon atoms and X is an amine or hydroxyl group, for example the DMRIE . In another embodiment, the cationic lipid can be associated with a neutral lipid, for example DOPE. Among these cationic lipids, preference is given to DMRIE (N- (2-hydroxyethyl) -N, N-dimethyl-2, 3-bis (tetradecyloxy) -1-? Ammonium ammonium; WO96 / 34109), advantageously associated with a neutral lipid, advantageously DOPE (dioleoyl-phosphatidyl-ethanol amine; Behr JP, 1994, Bioconjugate Chemistry, 5, 382-389), to form DMRIE-DOPE. Advantageously, the mixture of plasmid with the adjuvant is formed extemporaneously and advantageously in a contemporary manner with the administration of the preparation or in a short time before the administration of the preparation; for example, shortly before or before administration, the plasmid-adjuvant mixture is formed, advantageously to give enough time before the administration of the mixture to form a complex, for example, between about 10 to about 60 minutes before administration, such as about 30 minutes prior to administration. When DOPE is present, the molar ratio of DMRIE: DOPE is advantageously about 95: about 5 to about 5: about 95, more advantageously about 1: about 1, for example 1: 1. The weight ratio of DMRIE adjuvant or DMRIE-DOPE: plasmid can be between about 50: about 1 and about 1: about 10, such as about 10: about 1 and about 1: about 5, and advantageously about 1: about 1 and approximately 1: approximately 2, for example 1: 1 and 1: 2. In a specific embodiment, the pharmaceutical composition is administered directly in vivo, and the encoded product is expressed by the vector in the host. Methods of in vivo delivery of a vector encoding GHRH. (See, for example, U.S. Patent No. 6,423,693, EP Patent Publications No. 1052286, EP 1205551, U.S. Patent Publication 20040057941, WO 9905300 and Draghia-Akli et al., Mol Ther., 2002 December; 6 (6): 830-6, the descriptions of the which are incorporated herein by reference in their entireties) can be modified to deliver the canine GHRH of the present invention to a dog. The in vivo delivery of a vector encoding the canine GHRH described herein can be performed by an ordinary skill in the art given the teachings of the references mentioned in the above. Advantageously, the pharmaceutical and / or therapeutic compositions and / or formulations according to the invention comprise or consist essentially of, or consist of, an amount effective to induce a therapeutic response of one or more expression vectors and / or polypeptides as discussed in the present; and, an effective amount can be determined from this description, including the documents incorporated herein and the knowledge in the art, without undue experimentation. In the case of therapeutic and / or pharmaceutical compositions based on a plasmid vector, a dose may comprise, consist essentially of, or consist of, generally speaking, about 1 μg to about 2000 μg, advantageously about 50 μg to about 1000 μg and more advantageously from about 100 μg to about 800 μg of plasmid expressing GHRH. When therapeutic and / or pharmaceutical compositions based on a plasmid vector are administered With electroporation the doses of plasmids are generally between approximately 1.0 μg and 1 μg, advantageously between approximately 1 μg and 100 μg, advantageously between approximately 2 μg and 50 μg. The dose volumes may be between about 0.1 and about 2 ml, advantageously between about 0.2 and about 1 ml. These doses and dose volumes are suitable for the treatment of canines and other mammalian target species such as equines and felines. The therapeutic and / or pharmaceutical composition contains per dose from about 10 4 to about 10, advantageously from about 10 5 to about 10 10 and more advantageously from about 10 6 to about 10"viral particles of the recombinant adenovirus which expresses GHRH. therapeutic and / or pharmaceutical preparations based on a poxvirus, a dose can be between about 102 pfu and about 109 pfu.The pharmaceutical composition contains per dose from about 10: 1 to about 109, advantageously from about 10 6 to 10 pfu of poxvirus or recombinant herpesvirus expressing GHRH The dose volume of the compositions for target species that are mammalian, for example, the dose volume of canine compositions, based on viral vectors, for example compositions not based on viral vector poxvirus, is generally between about 0.1 to about 2.0 ml, preferably between about 0.1 to about 1.0 ml, and more preferably between about 0.5 ml to about 1.0 ml. It should be understood by one skilled in the art that the description herein is provided by way of example and the present invention is not limited thereto. From the description herein and knowledge in the art as the skilled person can determine the number of administrations, the route of administration, and the doses that are used for each injection protocol, without any undue experimentation. The present invention contemplates at least one administration to an animal of an efficient amount of a therapeutic composition made in accordance with the invention. The animal can be male, female, pregnant and newborn. This administration can be via several routes including, but not limited to, intramuscular (1M), intradermal (ID) or subcutaneous (SC) injection or via intranasal or oral administration. The therapeutic composition according to the invention can also be administered by an apparatus without needles (as, for example, with a Pigjet, Biojector or Vitajet apparatus (Bioject, Oregon, USA)). Another method for administering plasmid compositions is to use electroporation (see, for example, S. Tollefsen et al., Vaccine, 2002, 20, 3370-3378; S. Tollefsen et al., Scand. J. Immunol., 2003, 57, 229-238; S. Babiuk. et al., Vaccine, 2002, 20, 3399-3408; PCT Application No. WO99 / 01158). In another embodiment, the plasmid is delivered to an animal by a gene gun or bombardment of gold particles. In an advantageous embodiment, the animal is a vertebrate. In a more advantageous mode, the vertebrate is a dog. The present invention relates to the use of a viral vector encoding and expressing canine GHRH, mature canine GHRH, canine pre-proGHRH, canine proGHRH, variant, analog or fragment to produce a pharmaceutical composition for the treatment of a disease condition. For example, GH secretion stimulated by the administration of GHRH according to the present invention may have direct stimulatory effects on erythroid cells in an anemic animal. However, other disease conditions that may benefit from administration of a GHRH composition include, but are not limited to, cachexia, in particular, cachexia resulting from cancer, wound healing, bone healing, osteoporosis, obesity and the like. in a vertebrate. The canine GHRH expressing vector of the present invention has therapeutic utility in the treatment of cachexia (Bartlett et al., Cancer 1994, 73, 1499-1504 and Surgery 1995, 117, 260-267) in chronic diseases such as cancer, due to abnormalities of growth hormone production, in wound healing, bone healing , retardation of the aging process, osteoporosis and in anemia, (Sohmiya et al, J. Endocrinol, Invest 2000, 23, 31-36 and Clin Endocrinl, 2001, 55, 749-754). The intramuscular delivery of a plasmid encoding porcine GHRH has been shown to stimulate growth hormone and the release of IGF-I in dogs to treat anemia and cachexia (see, for example, Draghia-Akli et al., Mol Ther. 2002 December; 6 (6): 830-6). Chronic administration of GHRH has been shown to promote bone healing in dogs (see, for example, Dubreuil et al., Can J Vet Res. 1996 January; 60 (1): 7-13). It would be advantageous to administer the canine therapeutic GHRH vector of the present invention in place of the surgical polyethylene rod implant of Dubreuil et al. (Can J Vet Res. 1996 Jan; 60 (1): 7-13) to promote bone growth in an animal. Similarly, chronic administration of GHRH has been shown to promote the healing of. wounds in rats. { see, for example, Garre! and collaborators, Surg Res. 1991 October; 51 (4): 297-302). Again, it would be advantageous to administer the canine therapeutic GHRH vector of the present invention instead of the slow release pellet implant from Garrel et al. (Surg Res. 1991 October; 51 (4): 297-302) to promote wound healing in an animal. The invention also relates to a method for stimulating the immune response of a vertebrate. In one modality, the vertebrate is a bird, cat, cow, dog, fish, goat, horse, human, mouse, monkey, pig, rat or sheep. In a more advantageous mode, the vertebrate is a dog. Because the lymphocytes express GH-IGF-1, GHRH and their respective receptors, the administration of GHRH has been hypothesized to increase the function of the immune cell (see, for example, Khorram et al., J Clin Endocrinol Metab. 1997 November; 82 (11): 3590-6). In humans, administration of the GHRH analog to healthy elderly humans resulted in profound immuno-enhancing effects and may provide therapeutic benefits in the states of immunocompromised function (see, for example, Khorram et al., J Clin Endocrinol Metab 1997 November; 82 (11): 3590-6). The overexpression of GHRH in transgenic mice led to the significant stimulation of some parameters of immune function (see, for example, Diaiynas et al., 3 Clin Endocrino! Metab. 1997 November; 82 (11): 3590-6). In another mouse study, GHRH played a crucial role in the development of "experimental autoimmune encephalomyelitis and can provide the basis for a therapeutic procedure for the protection of autoimmune diseases (see, for example, Ikushima et al., J Immunol., 2003 September 15; 171 (6): 2769-72). Since the studies mentioned in the above suggest that GHRH enhances the function of the immune cell, administration of the therapeutic canine GHRH vector of the present invention would be advantageous for the stimulation of an immune response in an invertebrate. The invention will now be described further by means of the following non-limiting examples. EXAMPLES Example 1 The hypothalamus was taken from a dog and immediately frozen in liquid nitrogen. The total RNAs were extracted from the tissue using a QIAGEN kit (Rneasy Mini Protocol for Isolation of Total RNA Catalog No. 74104). The cells of the hypothalamus were dissociated with a Potter Dounce in 600 μl of denaturing solution of the equipment. This solution contained guanidinium isothiocyanate and beta-mercaptoethanol. The tissue homogenate was centrifuged for 5 minutes at 14000 RPM (rotations per minute) to remove cell debris. 600 μl of a 70% ethanol solution was added, the mixture was loaded onto a Rneasy column and centrifuged for 15 seconds at 10000 RPM. The column was rinsed twice with the buffer solution RW1 provided with the equipment. The RNA was exuded with 50 μl of RNAse-free buffer after centrifugation for 1 minute at 14,000 RPM. The cDNs were synthesized in 20 μl of the reaction mixture containing 5 mM MgCl 2, 20 mM Tris HCl pH 8.3, 100 mM KCl, 1 mM DTT, 1 M of each dNTP, 20 units of RNAse inhibitor, 50 units of Moloney murine leukemia virus reverse transcriptase, 2.5 μM random hexane nucleotide primers and 2 μl of total hypothalamic RNA. The reverse transcription step was performed with the following cycle: 23 ° C for 5 minutes, 42 ° C for 20 minutes, 99 ° C for 5 minutes and 10 ° C for 5 minutes. The accumulation of cDNAs was amplified by the Polymerase Chain Reaction (PCR) using the following oligonucleotides for the reaction: NB151 (18 mer) 5 '-ATGCYRCTCTGGGTGYTC-3' (SEQ ID NO: 5) NB152 (17 mer) 5 ' -TCATCCYTGGGAGTTCC-3 '(SEQ ID NO: 6) NB153 (21 mer) 5' -GCTACCGGAAGGTKCTGGGCC-3 '(SEQ ID NO: 7) NB154 (21 mer) 5' -GGCCCAGMACCTTCCGGTAGC-3 '(SEQ ID NO: 8) wherein Y is C or T, R is A or G, K is G or T, M is A or C. Oligonucleotides NB151 and NB154 were used to amplify a fragment of 136 base pairs (bp) corresponding to the 5 'end. of the GHRH gene. Oligonucleotides NB152 and NB153 were used to amplify a superimposed 206 bp fragment corresponding to the 3 'end of the GHRH gene. 100 μl of the reaction mixture contains 20 μl of the cDNA mixture, 2.5 units of DMA polymerase (J. Cline et al, Nucí Acid Res. 1996, 24, 3546-3551 and H. Hogrefe et al., Proc. Acad Sci. USA 2002, 99, 596-601), 50 mM Tris HCl pH 8.2, 0.5 μg of each oligonucleotide. { NB151 / N3154 or NB152 / NB153). The amplification was carried out with 35 cycles: 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute, followed at the end by an extension of 10 minutes at 72 ° C. In order to obtain the nucleotide sequence of the 5 'and 3' ends of the GHRH mRNA, the 5 'and 3' ends of the cDNAs were amplified according to a RACE protocol. For the 5 'end a mixture of 5 μi containing 3 μl of total hypothalamic RNA, 2 μM of primer oligo dt 5' -CDS, 2 μM of Smt oligonucleotide was heated to 70 ° C and cooled on ice for denaturation. A 10 μl reaction mixture containing 5 μl of the RNA denatured with oligonucleotides, 50 mM Tris HCl pH 8.3, 75 mM KCl, 6 mM MgCl 2, 2 mM DTT, 1 mM of each dNTP, 100 units of reverse transcriptase was incubated 90 minutes at 42 ° C and then diluted 1:10 in a buffer solution of 10 mM Tricine-KOH pH 8.5, 1 mM EDTA. An aliquot of 2.5 μl was used for the amplification in a 50 μl reaction mixture containing also 0.04 μM Universal primer, 0.2 μM oligonucleotide NB154, 40 mM Tricine-KOH pH 8.7, 15 mM KOAc, 3.5 mM Mg (OAc) 2, 3.75 μg / ml BASA, 0.005% Tween 20®, Nonidet-P40 at 0.005%, 0.2 μM of each dNTP, 2.5 units of Taq DNA polymerase. The amplification was carried out with 5 cycles that were carried out with 5 cycles of 3 minutes and 30 cycles of 94 ° C for 30 seconds, 65 ° C for 3 minutes, 72 ° C for 3 minutes. A fragment of 239 bp. Smt (30 mer) 5 '-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3' (SEQ ID NO: 9) Oligo dT 5 '-CDS 5' - (T) 25N-? N-3 ', where N_? is A, C or G and N is A, C, G or T. Universal Primer 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3 '(SEQ ID NO: 10). For the 3 'end the procedure is the same as described above except that the 5 μl mixture contains only 3 μl of SMART oligonucleotide (Clontech Laboratories, Palo Alto, California) and the 50 μl reaction mixture for PCR that contains aliquot of 2.5 μl of diluted cDNA, 0.04 μM of universal primer mixture, 0.2 μM of oligonucleotide NB153, 50 mM Tris HCl pH 8.3, 75 mM KCl, 6 mM MgCl2, 3.75 μg / ml BSA, 0.005 Tween 20® %, Nonodet-P40 at 0.005%, 0.2 μM each of dNTP, 2.5 units of Taq DNA polymerase. A fragment of 386 bp was obtained. SMART (57 mer) 5 '-AAGCAGTGGTATCAACGCAGAGTAC (T) 30N_aN-3' (SEQ ID NO: 11), where N_x is A, C or G and N or A, C, G or T. The fragments amplified by PCR are cloned directly into an appropriate plasmid. Colonies of E. coli were classified by hybridization of colony uplift. Plasmids with GHRH cDNA were identified using the NB155 probe labeled with alkaline phosphatase, corresponding to plasmids pCRII NB151 / 154 and pCRII NB152 / 153. NB155 (50 mer) 5'-GCCATCTTCACYAACARCTACCGGAAGGTBCTGGGCCAGCTVTCYGCCCG-3 '(SEQ ID NO: 12), where Y is C or T, R is A or G, B is C or G or T, V is A or C or G. The clones were sequenced. The nucleotide sequence encoding the canine pre-proGHRH comprising 321 bp as follows: 5 '-ATGCCACTCT GGGTGTTCTT CCTGGTGATC CTCACCCTCA GCAGTGGCTC CCACTCTTCC CCGCCATCCC TGCCCATCAG AATCCCTCGG TATGCAGACG CCATCTTCAC CAACAGCTAC CGGAAGGTGC TGGGCCAGCT GTCCGCCCGC AAGCTCCTGC AGGACATCAT GAGCCGGCAG CAGGGAGAGA GAAACCGGGA GCAAGGAGCA AAGGTACGAC TCGGCCGTCA GGTGGACAGT CTGTGGGCAA GCCAAAAGCA GATGGCATTG GAGAACATCC TGGCATCCCT GTTACAGAAA CGCAGGAACT CCCAAGGATG A-3 '(SEQ ID NO: 3).

The corresponding amino acid sequence is as follows: H-MPLWVFFLVI LTLSSGSHSS PPSLPIRIPR YADAIFTNSY RKVLGQLSAR KLLQDIMSRQ QGERNREQGA KVRLGRQVDS LWASQKQMAL ENILASLLQK RRNSQG-OH IDSE NO: 1). The signal peptide ranges from amino acid 1 to amino acid 20; mature GHRH ranges from amino acid 31 to amino acid 74. Example 2. The polynucleotide encoding canine GHRH is obtained by RT-PCR of total hypothalamic RNA using oligonucleotides NB184 and NB185 and DNA Polymerase (J.

Cline et al., 1996 and H. Hogrefe et al., 2002). NB184 (33 mer): 5'-TTTCGCGGATCCTATGCAGACGCCATCTTCACC-3 '(SEQ ID NO: 13) (contains a BamH I site at its 5' end). NB185 (35 mer): 5'-AAAGCTCTAGATCAACGGCCGAGTCGTACCTTTGC-3 '(SEQ ID NO: 14) (contains an Xba I site at its 5' end). The amplification is carried out with 1 cycle 'for the reverse transcription stage of 42 ° C for 15 minutes, 95 ° C for 5 minutes, 4 ° C for 5 minutes and 30 cycles for the PCR stage of 95 ° C for 45 seconds, 55 ° C for 45 seconds, 72 ° C for 1 minute. The PCR product (164 bp) is purified by extraction of phenol-chloroform and subsequently digested by BamH and Xba I to generate BamH I / Xba I of 147 bp (fragment A). The pABUO plasmid is derived from the plasmid pVR1020 (Vical Inc) by inserting a polylinker (BamH I, Not I, EcoR I, EcoR V, Xba I, Pml I, Pst I, Bgl II) corresponding to oligonucleotides PB326 and PB329 after digestion of BamH I / Bgl II of the plasmid. PB326 (40 mer) 5'-GATCTGCAGCACGTGTCTAGAGGATATCGAATTCGCGGCC-3 '(SEQ ID NO: 15) PB329 (40 mer) 5'- GATCCGCGGCCGCGAATTCGATATCCTCTAGACACGTGCT-3 (SEQ ID NO': 16) Plasmid pABUO is linearized by digestion of BamH I / Xba I to generate fragment B (5055 bp). Fragments A and B are subsequently purified and ligated to generate plasmid pNB179 (5202 bp) (FIG 1). Example 3: Plasmid pCRII NB151 / 154 is first digested by EcoRI. The EcoRI fragment (167 bp) is purified and finally digested by BsaWI to generate an EcoRI-BsaWI fragment (143 bp). Plasmid pCRII NB 152/153 is first digested by EcoRI. The EcoRI fragment (235 bp) is purified and finally digested by BsaWI to generate a BsaWI-EcoRI fragment (220 bp).

These two fragments are subsequently purified and cloned into linearized pCRII plasmid NB151 / 154 by EcoRI to generate the final plasmid pCRII NB151 / 152 (4295 bp). Example 4: The construction of pNB209 is based on a plasmid pVR1012 (Vical Inc.) encoding the canine GHRH precursor (106 amino acid polypeptide). The DNA fragment corresponding to the cGHRH gene, with the additional SalI and Xbal sites respectively, at the 5 'and 3' ends, is amplified by PCR using the primers NB361 and NB360, the plasmid pCRIINB151 / 152 as a template and the DNA polymerase . NB360 (30 mer): 5 '-AAAGCTCTAGATCATCCTTGGGAGTTCCTG-3' (SEQ ID NO: 17) (contains an Xbal site at its 5 'end) - NB361 (31 mer): 5' -TTTACGCGTCGACATGCTGCTCTGGGTGTTC-3 '(SEQ ID NO: 18) (contains a site I left at its 5 'end). The PCR fragment (345 bp) was purified by extraction of phenoxychloroform and subsequently digested by Sali and Xbal to generate fragment A (327 bp). Plasmid pVR1012 is linearized by Sall / Xbal digestion to generate fragment B (4880 bp). Fragments A and B are subsequently purified and ligated to generate plasmid? NB209 (5207 bp). FIG. 2 shows the plasmid map and the encoded ORF of pNB209. Example 5: Construction of plasmids pNB210 / cNE211 / pNB212 / pNB2I3: plasmids derived from pABUO containing modified GHRH genes, hybrid coding proteins containing the eukaryotic tPA guide peptide fused to the propeptide GHRH. The corresponding DNA fragment ai tPA (1-23 AA) with the additional SalI and Eco47III sites, respectively, at the 5 'and 3' ends, is obtained by PCR using the primers NB355 and NB356, the plasmid pABUO as a template and DNA polymerase (J. Cune et al., 1996 and H. Hogrefe et al., 2002). NB355 (20 mer): 5 '-CCGTCGTCGACAGAGCTGAG-3' (SEQ ID NO: 19) (contains a SalI site at its 5 'end). NB356 (24 mer) .: 5 '-AAAAGCGCTGGGCGAAACGAAGAC-3' (SEQ ID NO: 20) (contains an Eco47III site at the 5 'end). The PCR fragment (184 bp) is purified by extraction of ethanol-chloroform and subsequently directed by SalI and Eco47III to generate fragment A (172 bp). The DNA fragment corresponding to tPA (1-28) AA) with the additional Sali and Nael sites, respectively, the 5 'and 3' ends, is obtained by PCR using the primers NB355 and NB357, the plasmid pABUO as a template and DNA polymerase. NB355 (20 mer): 5 '-CCGTCGTCGACAGAGCTGAG-3' (SEQ ID NO: 19) (contains a SalI site at its 5 'end) - NB357 (42 mer): 5' -AAAGCCGGCATGGATTTCCTGGCTGGGCGAAACGAAGAC TGC-3 '(SEQ ID NO. : 21) (contains the additional 5 AA of the tPA guide peptide and a Nael site at the 5 'end). The PCR fragment (199 bp) is purified by extraction of phenol-chloroform and subsequently digested by Sali and Nael to generate fragment B (187 bp). The DNA fragment corresponding to the deletion (1-19 AA) GHRH with the additional NlalV and Xbal sites, respectively, the 5 'and 3' ends, is obtained by PCR using the primers MB358 and NB36Q, the plasmid pCRII NB151 / 152 as a template and DNA Polymerase. NB358 (21 mer): 5 '-TTTGGTTCCCCGCCATCCCTG-3' (SEQ ID NO: 22) (contains a NlalV site of the 5 'end). NB360 (30 mer): 5 '-AAAGCTCTAGATCATCCTTGGGAGTTCCTG-3' (SEQ ID NO: 17) (contains an Xbal site at its 5 'end). The PCR fragment (281 bp) is purified by the extraction of phenol-chloroform and subsequently NIalV and Xbal media is digested to generate fragment C (265 bp). The DNA fragment corresponding to the deletion (1-20 AA) of GHRH with the additional Stul and Xball sites at, respectively the 5 'and 3' ends, is obtained by PCR using the primers NB359 and NB360, the plasmid pCRII NB 151/15? as a template and DNA polymerase. NB359 (24 mer): 5 '-TTTAGGCCTCCATCCCTGCCCATC-3' (SEQ ID NO: 23) (contains a Stul site at its 5 'end). NB360 (30 mer): 5 '-AAAGCTCTAGATCATCCTTGGGAGTTCCTG-3' (SEQ ID NO: 17) (contains an Xbal site at its 5 'end). The PCR fragment (278 bp) is purified by extraction of phenol-chloroform and subsequently digested by Stul and Xbal to generate the D fragment (262 bp). The plasmid pVR1012 is linearized by Sall / Xbal digestion to generate the E fragment (4880 bp). The fragments?, A and C are subsequently purified and ligated to generate the plasmid pNB210 (5313 bp). FIG. 3 shows the plasmid map and the encoded ORF of pMB210. Fragments E, A and D are subsequently purified and ligated to generate the plasmid pNB211 (5310 bp). FIG. 4 shows the plasmid map and the encoded ORF of pNE211. The fragments E, B and C are subsequently purified and ligated to generate the plasmid pNB212 (5331 bp). FIG. 5 shows the plasmid map and the ORF encoded by pNB212. Fragments E, B and D are subsequently purified and ligated to generate the plasmid pNB213 (5328b). FIG. 6 shows the encoded ORF plasmid map of pNB213. Example 6: Construction of plasmids pNB214 / pNB215 The construction of plasmids pNB214 / pNB215: plasmids derived from pABUO containing modified GHRH genes, hybrid coding proteins containing human tPA fused to the mature peptide GHRH canine. The DNA fragment corresponding to the peptide (31-74 AA) GHRH with the additional Bstll07l and Xball sites at, respectively, the 5 'and 3' ends, is obtained by PCR using the primers NB362 and NB363, the plasmid pCRII NB151 / 152 as a template and DNA polymerase (J. Cline et al., 1996 and H. Hogrefe et al., 2002). NB362 (27 mer) :. 5 '-TTTGTATACGCAGACGCCATCTTCACC-3' (SEQ ID NO: 24) (contains the Bstll07I site at its 5 'end). NB363 (32 mer): 5'-AAAGCTCTAGATCAGAGTCGTACCTTTGCTCC-3 '(SEQ ID NO: 25) (contains the Xbal site at its 5' end). The PCR fragment (152 bp) is purified by extraction of phenol-chloroform and subsequently digests medial Bstll07II and Xbal to generate fragment F (136 bp). Fragments E, A and F are subsequently purified and ligated to generate the plasmid pNB214 (5184bp). FIG. 7 shows the map of the plasmid and the encoded ORF of pNB214. Fragment E, B and F are subsequently purified and ligated to generate the plasmid pNB215 (5202bp). FIG. 8 shows the plasmid map and the encoded ORF of pNB215. The fragments E, A and B are described in the example 17 Example 7: The canine pre-proGHRH nucleotide sequence is optimized by removing the cryptic splice sites, by adapting the .J. üi ction oe codon to one of Canis familiaris, by introducing the Kozak consensual sequence in order to improve expression. Such a modified gene is obtained by synthesis: SEQ ID NO: 40. The construction of pKB216: is based on a plasmid pVR1012 (Vical Inc.) which encodes the optimized canine GHRH precursor (106 amino acid polypeptide) SEQ ID MO: 40. Plasmid pCR-Script Amp-GHRH (Stratagene, Lajolla, CA, USA) that inserts the optimized canine pre-proGHRH gene is digested with Salí and Xbal to generate a Sall-Xbal fragment (336 bp) and the plasmid pVRl012 is linearized by a Sall / Xbal digestion to generate the E fragment (4880 bp). These two fragments are subsequently purified and ligated to generate the final plasmid pNB216 (5216 bp). Example 8: Construction of pNB217 / pNB218 plasmids: plasmids derived from pABUO containing modified GHRH genes, hybrid coding proteins containing the eukaryotic tPA guide peptide fused to the optimized GHRH propeptide. The DNA fragment corresponding to the suppression (1-20 AA) of GHRH optimized with the additional NlalV and Xbal sites at, respectively, the 5 'and 3' ends, is obtained by PCR using the primers NB364 and NB365, the plasmid pCR- Script Amp-GHRH as a template and DNA Polymerase. NB364 (24 mer): 5 '-TTTGGCCCCCCCAGCCTGCCCATC-3' (SEQ ID NO: 45) (contains a 5-fold Smal extreme site). NB365 (33 mer): 5'-AAAGCTCTAGATTATCAGCCCTGGCTGTTCCGC-3 '(SEQ ID NO: 46) (contains an Xbal site at its 5' end). The fragment of PCR (281 bp) is purified by the extraction of phenol-chloroform subsequently digested by NIalV and Xbal to generate the fragment G (265 bp). Plasmid pVR1012 is linearized by a Sall / Xbal digestion to generate the E fragment (4880 bp). The fragments E, A and G are subsequently purified and ligated to generate plasmid. pNB217 f I FIG. shows the plasmid map and the encoded ORF of pNB217. The fragments' E, B and G are subsequently purified and ligated to generate the plasmid pNB218 (5328bp). FIG. 11 shows the plasmid map and the encoded ORF of pNB218. The fragments A and B are described in example 5.

Example 9: Construction of pNB219 / pNB220 plasmids: plasmid derived from pABUO containing modified GHRH genes, coding hybrid proteins containing tPA fused to the mature peptide GHRH optimized dog. The DNA fragment corresponding to the peptide (31-74 AA) GHRH optimized with the additional Bstll07I and Xbal sites at, respectively, the 5 'and 3' ends, is obtained by PCR using the primers NB366 and NB367, the plasmid pCRII NB151 / 152 as a template and DNA polymerase (J. Cune et al., 1996 and H. Hogrefe et al., 2002). NB366 (27 mer): 5 '-TTTGTATACGCCGACGCCATCTTCACC-3' (SEQ ID NO: 47) (contains a Bstll07I site at the 5 'end).

NB367 (32 mer): 5 '-AAAGCTCTAGATCACAGCCGCACTTTGGCGCC-3' (SEQ ID NO: 48) (contains an Xbal site at its 5 'end). The PCR fragment (152 bp) is purified by extraction of phenol-chloroform and subsequently digested by Bstll07I and Xbal to generate the H fragment (136 bp). The E, A and H fragments are subsequently purified and ligated to generate the plasmid pNB219 (5184bp). FIG. 12 shows the plasmid map of the ORF encoded by pNB219. Fragments E, B and H are subsequently purified and ligated to generate the plasmid pNB220 (5199bp). FIG. 13 shows the plasmid map of the encoded ORF of pNB220. Fragments A and B are described in Example 5. Example 10: A method for low-voltage electroporation for DNA uptake and expression in an animal can be adapted from Example 2 of U.S. Patent Publication 20040057941, the description of which is incorporated by reference in its entirety. The method described herein can be modified to deliver the plasmid of the present invention without undue experimentation.

Injection of the direct intramuscular plasmid DNA followed by electroporation is a method for the local and controlled delivery of plasmid DNA into the skeletal muscle. This has the advantage that low amounts of plasmid (as low as 0.1 mg) are used, rather than the high amounts typically used with the passive delivery modalities. Although not wished to be related by theory, the mechanism of increased plasmid uptake by electroporation probably occurs through newly created membrane pores with or without active protein transport. Although it is not desired to be related by theory, the degree of permeabilization of muscle cells is dependent on the intensity of the electric field, the length of the pulses, the shape and type of electrodes (Bureau et al., Biochim Biophys Acta. 2000 May 1; 1474 (3): 353-9 and Gilbert et al., Biochim Biophys Acta. 1997 February 11; 1334 (1): 9-14), and cell size (Somiari et al., Mol Ther. September; 2 (3): 178-87). The classical electrode configuration, the plates or a pair of wire electrodes placed 4 mm apart were shown to be effective in rodents, but in large mammals such as pigs or humans the increased skin resistance, the thickness of the subcutaneous fat tissue and the problem for tissue damage without the intensity of the electric field would be proportionally increased, makes these types of electrodes impractical. Porcine or dog muscle fibers are very large and consequently more suitable for electroperbilization than the surrounding muscle. In this report, the inventors show that a single injection of several dosages of GHRH or analogous nucleic acid sequences followed by electroporation with intramuscular applicators, in a large mammal is sufficient to produce therapeutic plasma hormone levels, with biologically significant effects They can treat anemia, reverse waste, allow the echo to gain weight and prolong the life expectancy of the chronic patient. The pSP-HV-GHRH system (a porcine GHRH expressing vector) F supplied the anterior muscle of the left tibia of healthy dogs via an in vivo electroporation. A group of 4 dogs (2 males and 2 females) were used co or controls and 3 groups of 8 dogs (4 males and 4 females) were injected with the pSP-HV-GHRH system. The dogs were injected with single vehicles (control) or 200 mcg, or 600 mcg or 1000 mcg of pSP-HV-GHRH followed by needle electroporation. An indication of the increased systemic levels of GHRH and GH is an increase in serum IGF-I concentration. Therefore, after 28 days post-injection the blood serum was collected from the dogs that were injected with the serum vehicle (control), or 200 mcg, or 600 mcg or 1000 mcg of pSP-HV-GHRH and the IGF-I levels were determined. IGF-I levels for dogs injected with 600 mcg were 3 times higher than control treated animals (vehicle only) (FIG 2). The increase in IGF-I levels was statistically significant (p <0.046). Although animals injected with 200 mcg and 1000 mcg of plasmid showed higher IGF-I levels than controls, IGF-I levels were lower than in animals injected with 600 mcg. The increased IGF-I levels corresponding to higher GHRH levels are in agreement with other studies that used recombinant porcine GH ("pGH") in dogs. For example, there were increased dose-related serum IGF-I levels (approximately 2-10 times) that correlated with elevated serum GH levels in dogs treated with pGH. Although it is not desired to be related by theory, the hormone of growth hormone release (GHRH) stimulates the production and release of the anterior pituitary of growth hormone (GH), which in turn stimulates the production of IGF- I of the liver and other target organs. Thus, an indication of increased systemic levels of GHRH and GH is an increase in the concentration of IGF-I in the serum. The level of IGF-I in the serum in healthy dogs injected with 200, 600 and 1000 mcg of pSP-HV-GHRH were all higher 28 days after injection when compared with the pre-injection values. Dogs injected with 600 mcg of pSP-HV-GHRH showed the highest statistically significant increase (eg, greater than 90%, p <0.046) in IGF-I levels, indicating that 600 mcg may be the concentration optimal for healthy dogs. Example 11: Construction of plasmids pNB240, pNB239 and pNB244. The construction of pNB240 is based on a plasmid pVR1012 (Vical Inc.) comprising a modified GHRH gene, which encodes a hybrid protein containing the pre-pro-GHRH deleted in the C-terminal propeptide and fused to the epitope tag c -myc. The constructs of pNB239 and pNB244 are based on a plasmid pVR1012 (Vicaí Inc.) comprising a modified GHRH gene, which encodes a hybrid protein containing the equine IGF1 (Equine Insulin-like Growth Factor 1) peptide signal sequence (SSIGFl , disclosed in Genbank under accession numbers U28070 (24-48 AA) and U85272) fused to the modified GHRH and fused to the c-myc epitope tag. The DNA fragment encoding the pre-proGHRH deleted in the C-terminal propeptide (corresponding to 1-74 AA) and fused to the c-myc epitope tag, with the additional Xbal site at the 5 'end is amplified by PCR using primers PB091 and NB 12, plasmid pNB209 (see, example 4) as a template and the DNA Polymerase. PB091 (22 mer): 5 '-CCAGACATAATAGCTGACAGAC-3' (SEQ ID NO: 51). NB412 (68 mer): 5'-AAAGCTCTAGATTAGAGATCCTCTTCTGAGATAAGCTTTTGTTCGAGTCGTACCTTTGCTC CTTGCTC-3 '(contains the c-myc epitope tag and an Xbal site at its 3' end) (SEQ ID NO: 52). The PCR fragment (338 bp) was purified by extraction of phenol-chloroform and subsequently digested by Sali and Xbal to generate fragment A (261 bp). The equine IGF1 pre-pro nucleotide sequence is optimized by removing the cryptic splice sites, by adapting codon usage, by introducing a Kozak consensual sequence before the start codon in order to improve expression. Such a modified gene is obtained from the synthesis: SEQ ID NO: 65. The pCR-Script Amp-elGFI plasmid (Stratagene, Lajolla, CA, USA) comprising the optimized equine IGF1 gene is used as a template for PCR with the NB393 primers and NB394 and DNA Polymerase. NB393 (38 mer): 5'- TTIGCGTCGACGCCACCATGCACATCATGAGCAGCAGC-3 '(contains a site I left at its 5' end) (SEQ ID NO: 66). NB394 (38 mer): 5'-AAAGCTCTAGATTATCACATCCGGTAGTTCTTGTTGCC-3 '(contains an Xbal site at its 5' end) (SEQ ID NO: 67). The PCR fragment (424 bp) is purified by extraction. of phenol-chloroform and subsequently digested by Sali and Xbal to generate the F fragment (408 bp). The plasmid pVR1012 is linearized by Sall / Xbal digestion to generate the E fragment (4880 bp). Fragments E and F are subsequently purified and ligated to generate the plasmid pNB231 (5288bp). The DNA fragment corresponding to the equine SSIFG1 (1-25 AA of FIGS. 15-18), with the additional Nael site at the 3 'end, is obtained by PCR using the primers PB091 and NB398, plasmid pNB231 as a template and the DNA Polimeras.a (J. Cline et al., 1996 and H. Hogrefe et al., 2002). PB091 (22 mer) (SEQ ID NO: 51). NB398 (25 mer): 5 '-AAAGCCGGCGGTGGCGCTGCTGGTG-3' (contains a Nael site at its 3 'end) (SEQ ID NO: 53). The PCR fragment (159 bp) is purified by extraction of phenol-chloroform and subsequently digested by Sali and Nael to generate fragment B (86 bp). The DNA fragment corresponding to the mature peptide GHRH (31-74 AA) fused to the c-myc epitope tag with the additional Bstll07I and Xbal sites at, respectively, the 5 'and 3' ends, is obtained by PCR using the primers NB362 and NB412, the plasmid pNB209 as a template and the DNA Polymerase. NB362 (27 mer) (SEQ ID NO: 24). NB412 (68 mer) (SEQ ID NO: 52). The PCR fragment (182 bp) is purified by extraction of phenol-chloroform and subsequently digested by Bstll07I and Xbal to generate fragment C (166 bp). The DNA fragment corresponding to the GHRH propeptide deleted from the C-terminal propeptide (20-74 AA) fused to the c-myc epitope tag, with the additional NIalV and Xbal sites at, respectively, the 5 'and 3' ends are obtained by PCR using the primers NB358 and NB412, the plasmid pNB209 as a template and the DNA Polymerase. NB358 (21 mer) (SEQ ID NO: 22). NB412 (68 mer) (SEQ ID NO: 52). The PCR fragment (215 bp) is purified by extraction of phenol-chloroform and subsequently digested by Bstll07I and Xbal to generate fragment D (199 bp).

Plasmid pVR1012 is linearized by Sall / Xbal digestion to generate E fragment (4880 bp). Fragments E and A are subsequently purified and ligated to generate the plasmid pNB240 (5141bp). FIG. 14 shows the plasmid map and the ORF encoded by pNB240. The fragments E, B and C are subsequently purified and ligated to generate the plasmid pNB239 (5132bp). FIG. 15 shows the plasmid map and the encoded ORF of pNB239. The fragments E, B and D are subsequently purified and ligated to generate the plasmid pNB244 (5165bp). FIG. 16 shows the plasmid map and the encoded ORF of pNB244. In vitro expressions of the fusion proteins encoded by pNB239, pNB240 and pNB244 were studied after transient transfection of CHO-Kl cells (Chinese hamster ovary cells, available before the Collection American Culture Type ATCC Cat. No. CCI-61), using Lipofectamin 2000. CHO-Kl cells at 90% confluence in 6 cm diameter plates were transfected with 5 μg of plasmid and 10 μl of lipofectamine each one, according to the manufacturer's instructions. After transfection, the cells were cultured in the medium MEM-glutamax containing 1% FCV (fetal sheep serum) for 24 hours. The culture supernatants are collected and concentrated 40 times by precipitation with protein trichloroacetic acid. The cells were washed with PBS (phosphate buffered saline), harvested by scraping and used using the Laemmli SDS-PAGE charge buffer. The production of recombinant protein and secretion were analyzed by submitting the extracts whole cells and the concentrated culture supernatants (40 x) to SDS-PAGE and western blotting, using an anti-c-myc monoclonal antibody (Euromedex, France). Plasmid pNB240 encodes a hybrid protein containing amino acids 1 to 74 of preproGHRH and fused to the c-myc epitope tag. Plasmid pNB239 encodes a hybrid protein containing the equine IGF1 peptide signal sequence (SSIGF1) fused to the mature GHRH peptide (31-74 AA) and the c-myc epitope tag. Plasmid pNB244 encodes a hybrid protein containing the equine IGF1 peptide signal sequence (SSIGF1) fused to the propeptide GHRH (20-74 AA) and the c-myc epitope tag. The results of the expression are summarized in the Table 1. All three constructions lead to a 6.3 kDa expression product in the culture supernatants, which show the correct maturation of the hormones recombinants, Table 1: Example 12: Construction of plasmids pNB232 and pNB245. The constructs of pNB232 and pNB245 are based on a plasmid pVR1012 (Vical Inc.) comprising a modified GHRH gene, which modifies a hybrid protein encoding the equine IGF1 peptide sequence signal (SSIGF1, disclosed in Genbank under accession numbers U28070 (24-48 AA) and U85272) fused to the modified GHRH. The DNA fragment corresponding to the propeptide GHRH deleted from the N-terminal propeptide (corresponding to 31-106 AA) with the additional Bstl sites 1071 and Xbal at, respectively, the 5 'and 3' ends, is obtained by PCR using the primers NB362 and NB421, the plasmid pNB209 ( see example 4) as a template and DNA polymerase (J. Cune et al., 1996 and H. Hogrefe et al., 2002). NB362 (27 mer) (SEQ ID NO: 24) NB421 (36 mer): '-AAAGCTCTAGATTATCCTTGGGAGTTCCTGCGTTTC-3' (contains an Xbal site at its 5 'end) (SEQ ID NO: 54), The PCR fragment (248 bp) is purified by extraction with phenol-chloroform and subsequently digested by Bstl 1071 and Xbal to generate the G fragment (232 bp). The plasmid pVR1012 is linearized by a Sall / Xbal digestion to create the H fragment (4880 bp). Fragments H, B and F are subsequently purified and ligated to generate the plasmid pNB232 (5102bp). FIGURE 17 shows the plasmid map of the ORF encoded by pNB232. The H fragments, B and G are subsequently purified and ligated to generate the plasmid pNB245 (5198bp). FIGURE 18 shows the plasmid map of the encoded ORF of pNB245. Fragment B is described in example 11. Fragment F is described in example 6. Example 13: Construction of plasmids pNB228, pNB297 and pNB298 Plasmid pNB228 corresponds to the main strand of plasmid pVR1012 (Vical Inc.) containing the nucleic acid fragment encoding the preproGHRH peptide deleted from the carboxy-terminal propeptide. Plasmid pNB297 corresponds to the plasmid backbone pVR1012 (Vical Inc.) containing the nucleic acid fragment encoding amino acids 1 to 74 of preproGHRH and a glycine at its carboxyterminal end. The carboxy-terminal glycine must be easily amidated by the amidation enzyme. Plasmid pNB298 corresponds to the 'plasmid backbone pVRl012 (Vical Inc.) containing the nucleic acid fragment encoded by the equine IGF1 signal sequence (SSIGF1) fused to mature GHRH (31-74 AA) and a glycine in its carboxy-terminal end. The DNA fragment corresponding to the preproGHRH deleted in the carboxy-terminal propeptide, with the additional SalI and Xbal sites at the 5 'and 3' ends, respectively, is amplified by PCR using the primers NB361 and NB363, the plasmid pNB209 as a template DNA polymerase NB361 (31 mer): 5 '-TTTACGCGTCGACATGCTGCTCTGGGTGTTC-3' (SEQ ID NO: 17) (contains a SalI site at its 5 'end) NB363 (32 mer): 5' -AAAGCTCTAGATCAGAGTCGTACCTTTGCTCC-3 '(SEQ ID NO: 24 ) (contains an Xbal site at its 5 'end) The PCR fragment (249 bp) is purified by extraction with phenol-chloroform and subsequently digested by Sali and Xbal to generate fragment A (231 bp). The DNA fragment corresponding to preproGHRH (1-74 AA) and a glycine at its carboxy-terminal end, with the additional Salí and Xbal sites at the 5 'and 3' ends, respectively, is amplified by PCR using the primers NB361 and NB476, the plasmid pNB209 as a template and the DNA polymerase. NB361 (31 mer): 5 '-TTTACGCGTCGACATGCTGCTCTGGGTGTTC- 3' (SEQ ID NO: 17) (contains a SalI site at its 5 'end) NB476 (32 mer): 5 '-AAAGCTCTAGATTAGCCGAGTCGTACCTTTGC-3' (SEQ ID NO: 68) (contains an Xbal site at its 5 'end) The PCR fragment (252 bp) is purified by extraction with phenol-chloroform and subsequently digested by Sali and Xbal to generate fragment B (234 bp). The DNA fragment corresponding to the equine IGF1 signal sequence (SSIGF1) fused to the mature GHRH (31-74 AA) and a glycine at its carboxy-terminal end, with the additional SalI and Xbal sites at the 5 'and 3' ends , respectively, is obtained by PCR using the primers NB393 and NB476, the plasmid pNB232 as a template and the DNA polymerase (J. Cline et al., 1996 and H. Hogrefe et al., 2002). NB393 (38 mer): 5'-TTTGCGTCGACGCCACCATGCACATCATGAGCAGCAGC -3 '(SEQ ID NO: 66) (contains a SalI site at its 5' end) NB476 (32 mer): 5 '-AAAGCTCTAGATTAGCCGAGTCGTACCTTTGC-3' (SEQ ID NO: 68 ) (contains an Xbal site at its extreme 5 ') The PCR fragment (241 bp) is purified by extraction of phenol-chloroform and subsequently digested by Sali and Xbal to generate fragment C (225 bp). Plasmid pVR1012 is linearized by Sall / Xbal digestion to generate fragment E (4880 bp). The E and A fragments are subsequently purified and ligated to generate the plasmid pNB228 (5111 bp). FIG. 19 shows the plasmid map and the encoded ORF of pNB228. Fragments E and B are subsequently purified and ligated to generate the plasmid pNB297 (5114 bp). FIG. shows the plasmid map and the encoded ORF of pNB297. Fragments E and C are subsequently purified and ligated to generate the plasmid pNB298 (5105 bp). FIG. 21 shows the plasmid map and the encoded ORF of pNB298. Plasmids similar to plasmids pNB297 and pNB298 containing porcine GHRH (SEQ ID NO: 82) in place of the canine GHRH sequence according to the method described in example 13 and examples 6, 11, 12 using the following primers , are constructed: NB464 (37 mer): 5'-TTTACGCGTCGACATGCTGCTGTGGGTGTTCTTCCTG -3 '(SEQ ID NO: 83) corresponding to primer NB361 and NB465 (39 mer): 5'-TTTTGCTCTAGATTAGCCCAGCCTCACCCTGGCGCCCTG -3' (SEQ ID NO: 84) corresponding to primer NB476 NB416 (27 mer): 5 '-TTTGTATACGCCGACGCCATCTTCACC -3' (SEQ ID NO: 85) corresponding to primer NB362 and NB418 (38 mer): 5'-AAAGCTCTAGATTACAGCCTCACCCTGGCGCCCTGCTC-3 '(SEQ ID NO: 86) corresponding to primer NB363 NB465 (39 mer) (SEQ ID NO: 84) corresponding to primer NB476. Porcine GHRH (231 mer) (SEQ ID NO: 82): 5'-ATGCTGCTGTGGGTGTTCTTCCTGGTGACCCTGACCCTGAGCAGCGGCAG CCTGAGCAGCCTGCCCAGCCAGCCCCTGAGGATGCCCAGGTACGCCGACG CCATCTTCACCAACAGCTACAGGAAGGTGCTGGGCCAGCTGAGCGCCAGG AAGCTGCTGCAGGACATCATGAGCAGGCAGCAGGGCGAGAGGAACCAGGA GCAGGGCGCCAGGGTGAGGCTGGGCAGGCAGGTGGACAGCATGTGGGCCG ACCAGAAGCAGATGGCCCTGGAGAGCATCCTGGCCACCCTGCTGCAGGAG CACAGGAACAGCCAGGGCTGA-3 '. Example 14: Construction of plasmids pNB299 and pNB300 Plasmid pNB299 corresponds to the main strand of plasmid pVR1012 (Vical Inc.) which contains the nucleic acid fragment encoding the equine IGF1 signal sequence (SSIGF1) fused to mature GHRH (31-74 AA). Plasmid pNB300 corresponds to the plasmid backbone pVR1012 (Vical Inc.) which contains the nucleic acid fragment encoding the sequence of Equine IGF1 signal (SSIGF1) fused to mature GHRH (amino acids 31-74 of the precursor) and canine serum albumin (amino acids 25 to 608 of the precursor) through a tetrapeptide linker (GSGS). The DNA fragment corresponding to the Equine IGF1 signal sequence (SSIGF1) fused to the mature GHRH (31-74 AA), with the additional SalI and PshAI sites at, respectively, the 5 'and 3' ends, is obtained by PCR using the primers NB393 and NB477, the plasmid pNB232 as a template and the DNA polymerase. NB393 (38 mer): 5'-TTTGCGTCGACGCCACCATGCACATCATGAGCAGCAGC -3 '(SEQ ID NO: 17) (contains a SalI site at its 5' end) NB477 (50 mer): 5'-AAATCTAGAGCCGGTTTAAAAGACCGTAGTCGTACCTTTGCTCCTTGCTC -3 '(SEQ ID NO: 75 ) (contains the Xbal and PshAI sites at its 5 'end) The PCR fragment (250 bp) is purified by extraction with phenol-chloroform and subsequently digested by Sali and Xbal to generate fragment A (236 bp). Plasmid pVR1012 is linearized by Sall / Xbal digestion to generate fragment E (4880 bp). Fragments E and A are subsequently purified and ligated to generate the plasmid pNB299 (5116bp). The FIGURE 22 shows the plasmid map and the encoded ORF of pNB299. The DNA fragment corresponding to the GSGS linker fused to canine serum albumin (amino acids 25 to 608), which additional Nael and Xbal sites in, respectively, the 5 'and 3' ends, is obtained by RT-PCR. The liver is collected from a dog and immediately frozen in liquid nitrogen. The total RNAs are extracted from the tissue using a QIAGEN kit (Protocol for the Isolation of RNA Total Rneasy Mini Catalog ref. 74104).

Liver cells dissociate with a Potter Dounce in 600 μl of denaturing solution of the equipment. This solution contains guanidinium isothiocyanate and beta-mercaptoethanol. The tissue homogenate is centrifuged at 5 minutes at 14,000 RPM (rotations per minute) to remove cell debris. 600 μl of a 70% ethanol solution is added and the mixture is loaded on a Rneasy column and centrifuged 15 seconds at 10000 RPM. The column is rinsed twice with the regulatory solution RWl provided with the equipment. The RNA is eluted with 50 μl of RNase-free buffer after centrifugation for 1 minute at 14,000 RPM. The cDNAs are synthesized in a 20 μl reaction mixture containing 5 mM MgC12, 20 mM Tris HCl, pH 8.3, 100 mM KCl, 1 mM DTT, 1 mM of each dNTP, 20 RNAse inhibitor units, 50 Moloney murine leukemia virus reverse transcriptase units, 2.5 μM random hexane nucleotide primers and 2 μl of total liver RNA. The reverse transcription stage is carried out with the following cycle: 23 ° C for 5 minutes, 42 ° C for 20 minutes, 99 ° C for 5 minutes and 10 ° C for 5 minutes. The accumulation of cDNAs is amplified by the Polymerase Chain Reaction (PCR) using the DNA polymerase and the following oligonucleotides for the reaction: NB478 (43 mer): 5'- TTTGCCGGCTCAGGATCCGAAGCATATAAGAGTGAGATTGCTC -3 '(SEQ ID NO: 78) (contains a Nael site and the GSGS linker at its 5 'end) y) NB479 (35 mer): 5'-AAAGCTCTAGATTAGACTAAGGCAGCTTGAGCAGC -3' (SEQ ID NO: 79) (contains an Xbal site at its 5 'end) The PCR fragment ( 1784 bp) is purified by extradition with phenol-chloroform and subsequently digested by Nael and Xbal to generate fragment B (1768 bp). Plasmid pNB299 is linearized by digestion of PshAI / Xbal to generate fragment C (5098 bp). Fragments C and B are subsequently purified and ligated to generate the plasmid pNB300 (6866 bp). The FIGURE 23 shows the plasmid map and encoded ORF of pNB300. Plasmids similar to the plasmids pNB299 and pNB300 containing the porcine GHRH (SEQ ID NO: 82) instead of the canine GHRH sequence according to the method described in the present example 14 using the following primers, are constructed: NB459 (53 mer ): 5'-AAATCTAGAGCCGGTTTAAAAGACCGTAGTCTCACCCTGGCGCCCTGCTCCTG-3 '(SEQ ID NO: 87) corresponding to primer NB477. NB457 (40 mer): 5'-TTTGCCGGCTCAGGATCCGATACATACAAGAGTGAAATTG-3 '(SEQ ID NO: 88) corresponding to primer NB478 and NB458 (29 mer): 5' -AAAGCTCTAGATTAGGCTAAGATCCCTCG-3 '(SEQ ID NO: 89) corresponding to primer NB479. Having described in this manner in detail the advantageous embodiments of the present invention, it is to be understood that the invention defined by the preceding paragraphs is not going to be limited to the particular details set forth in the foregoing description since many obvious variations thereof are possible without departing from the spirit or scope of the present invention.

Claims (40)

1. CLAIMS 1. An expression vector, characterized in that it comprises a polynucleotide encoding a canine proGHRH deleted from the N-terminal propeptide, wherein the polynucleotide comprises a peptide signal sequence fused to the nucleotide base sequence from nucleotide 91 to nucleotide 321 of SEQ ID NO: 3.
2. 2. An expression vector, characterized in that it comprises a polynucleotide encoding a mature canine GHRH, wherein the polynucleotide comprises a peptide signal sequence fused to the nucleotide base sequence of SEQ ID NO. NO: 4. The expression vector according to claim 1, characterized in that the peptide signal sequence fused to the nucleotide base sequence is operatively linked to an enhancer and / or a promoter or wherein the sequence of signal of the peptide is an equine IGF-1 signal peptide or wherein the peptide signal sequence fused to the base sequence of nucleotides is operably linked to an enhancer and / or a promoter and wherein the peptide signal sequence is an equine IGF-1 signal peptide. 4. The expression vector according to claim 2, characterized in that the peptide signal sequence fused to the nucleotide base sequence is operably linked to an enhancer and / or a promoter wherein the signal sequence of the peptide is an equine IGF-1 signal peptide or wherein the peptide signal sequence fused to the nucleotide base sequence is operably linked to a enhancer and / or a promoter and wherein the peptide signal sequence is an equine IGF-1 signal peptide. 5. A formulation for delivery and expression of a canine proGHRH deleted from the N-terminal propeptide in a cell, characterized in that the formulation comprises the vector of claim 1 and a pharmaceutically or veterinarily acceptable carrier, vehicle or excipient. 6. A formulation for delivery and expression of a mature canine GHRH in a cell, characterized in that the formulation comprises the vector of claim 2 and a pharmaceutically or veterinarily acceptable carrier, vehicle or excipient. 7. The formulation according to claim 5, characterized in that the carrier, vehicle or excipient facilitates transfection and / or improves the preservation of the vector or protein. 8. The formulation according to claim 6, characterized in that the carrier, vehicle or excipient facilitates transfection and / or improves the preservation of the vector or protein. 9. A method for delivering GHRH to a vertebrate, characterized in that it comprises injecting the formulation of claim 5 into the vertebrate. 10. A method for delivering GHRH to a vertebrate, characterized in that it comprises injecting the formulation of claim 6 into the vertebrate. 11. The method according to claim 9, characterized in that the vertebrate is a dog. 12. The method according to claim 10, characterized in that the vertebrate is a dog. 13. A method for treating anemia, bone healing, cachexia, obesity, osteoporosis or wound healing in a vertebrate, characterized in that it comprises administering an effective amount of the formulation of the claim 5 to the vertebrate cells and expressing GHRH in the cells 14. A method for treating anemia, bone healing, cachexia, obesity, osteoporosis or wound healing in a vertebrate, characterized in that it comprises administering an effective amount of the formulation of claim 6 to the vertebrate cells and expressing GHRH in the cells . 15. The method according to claim 13, characterized in that the vertebrate is a dog. 16. The method of compliance with the claim 14, characterized in that the vertebrate is a dog. 17. A method for stimulating an immune response in a vertebrate, characterized in that it comprises administering an effective amount of the formulation of claim 5 to the vertebrate cells and expressing GHRH in the cells. 18. A method to stimulate an immune response in a vertebrate, characterized in that it comprises administering an effective amount of the formulation of claim 6 to the vertebrate cells and expressing GHRH in the cells. 19. The method according to claim 17, characterized in that the vertebrate is a dog. 20. The method according to claim 18, characterized in that the vertebrate is a dog. 21. An expression vector, characterized in that it comprises a polynucleotide encoding a proGHRH deleted from the N-terminal propeptide, wherein the polynucleotide comprises an equine IGF-1 signal peptide operably linked to an enhancer and / or a promoter. 22. An expression vector, characterized in that it comprises a polynucleotide encoding a mature GHRH, wherein the polynucleotide comprises an equine IGF-1 signal peptide operably linked to an enhancer and / or a promoter. 23. The expression vector according to claim 21, characterized in that the proGHRH comprises an additional glycine at the C-terminus. 24. The expression vector according to claim 22, characterized in that the GHRH comprises a bound serum albumin or wherein the GHRH comprises a serum albumin linked through a tetrapeptide linker. 25. A formulation for delivery and expression of a canine proGHRH deleted from the N-terminal propeptide in a cell, characterized in that the formulation comprises the vector of claim 21 and a pharmaceutically or veterinarily acceptable carrier, vehicle or excipient. 26. A formulation for delivery and expression of a mature GHRH in a cell, characterized in that the formulation comprises the vector of claim 22 and a pharmaceutically or veterinarily acceptable carrier, vehicle or excipient. 27. The formulation according to claim 25, characterized in that the carrier, vehicle or excipient facilitates transfection and / or improves the preservation of the vector or protein. 28. The formulation according to claim 26, characterized in that the carrier, vehicle or excipient facilitates transfection and / or improves the preservation of the vector or protein. 29. A method for delivering GHRH to a vertebrate, characterized in that it comprises injecting the formulation of claim 25 into the vertebrate. 30. A method for delivering GHRH to a vertebrate, characterized in that they comprise injecting the formulation of claim 26 into the vertebrate. 31. The method of compliance with the claim 29, characterized in that the vertebrate is cattle, dog or pig. 32. The method of compliance with the claim 30, characterized in that the vertebrate is cattle, dog or pig. 33. A method for treating anemia, bone healing, cachexia, obesity, osteoporosis or wound healing in a vertebrate, characterized in that it comprises administering an effective amount of the formulation of claim 25 to the vertebrate cells and expressing GHRH in the cells . 34. A method for treating anemia, bone healing, cachexia, obesity, osteoporosis or wound healing in a vertebrate, characterized in that it comprises administering an effective amount of the formulation of claim 26 to the cells of the vertebrate and expressing GHRH in the cells . 35. The method according to claim 33, characterized in that the vertebrate is cattle, dog or pig. 36. The method according to claim 34, characterized in that the vertebrate is cattle, dog or pig. 37. A method for stimulating an immune response in a vertebrate, characterized in that it comprises administering an effective amount of the formulation of claim 25 to the vertebrate cells and expressing GHRH in the cells. 38. A method for stimulating an immune response in a vertebrate, characterized in that it comprises administering an effective amount of the formulation of claim 26 to the cells of the vertebrate and expressing GHRH in the cells. 39. The method of compliance with the claim 37, characterized in that the vertebrate is cattle, dog or pig. 40. The method of compliance with the claim 38, characterized in that the vertebrate is cattle, dog or pig.