HUE033580T2 - Canine ghrh gene, polypeptides and methods of use - Google Patents

Description

(12) Date of publication and mention (51) Int Cl .: of the grant of the patent: C07K 14l6O <2006 01> 05.04.2017 Bulletin 2017/14 (86) International application number: (21) ) Application number: 05752603.0 PCT / US2005 / 015522 (22) Date of filing: 02.05.2005 (87) International publication number: WO 2005/085448 (15.09.2005 Gazette 2005/37)

(54) CANINE GHRH GENE, POLYPEPTIDES AND METHODS OF USE

GHRH GEN VOM HUNDE, POLYPEPTIDE UND METHODEN ZU DEREN GEBRAUCH GENE GHRH CANIN, SES POLYPEPTIDES ET SES METHODS D'UTILIZATION (84) Designated Contracting States: (72) Inventors: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR · FISHER, Laurent, Bernard HU IE IS IT LI LT LU MC NL PL PT RO SE SI SKTR F-69110, Sainte Foy Les Lyon (FR)

Designated Extension States: · CACHET, Nathalie, Michele AL BA HR EN MC YU F-69100, Sainte Foy Les Lyon (FR) • BARZU-LE-ROUX, Simona (30) Priority: 03.05.2004 US 838122 F-69210 Lentilly ( FR) 17.12.2004 US 15461 (74) Representative: D Young &amp; Co LLP (43) Date of publication of application: 120 Holborn 10.01.2007 Bulletin 2007/02 London EC1N 2DY (GB) (73) Proprietor: Merial, Inc.

Georgia 30096 (US)

Note: Within a period of nine months from the date of publication of the publication of the European Patent Office of the Implementing Regulations. Notice of opposition to the opposition has been paid. (Art. 99 (1) European Patent Convention).

Description [0001] This application claims priority from U.S. Application Serial No. 11 / 015,461 filed December 17,2004 which claims priority from U.S. Application Serial No. 10 / 838,122 filed May 3, 2004 which claims priority from U.S. Provisional Application Serial No. 60 / 467,405, filed May 1,2003. And,. \ Tthe documents relating to the foregoing applications,. \ T cited or referenced in this document, description, product specifications, and product sheets for the product of the invention.

FIELD OF THE INVENTION

The present description or to a canine pre-proGHRH polypeptide, canine mature GHRH peptide, is an isolated polynucleotide which encodes the canine pre-proGHRH or the canine mature GHRH.

[0003] In the anchor the description is a vector encoding and expressing pre-proGHRH or GHRH. Such a method can be used to treat disease and growth hormone deficiency, in particular in dogs.

In another embodiment, the present invention provides a method for delivering the peptide GHRH to a vertebrate, in particular to a dog, in a canine pre-proGHRH or a canine GHRH to the animal host.

The description and method of treatment of anemia, cachexia, wound healing, bone healing, osteoporosis, obesity in vertebrates, in particular in dog.

BACKGROUND OF THE INVENTION

Growth hormone-releasing hormone (GHRH) is a hypothalamic peptide that plays a critical role in controlling the hormone (GH) by the anterior pituitary. Human GHRH is also a C-terminal amidated peptide of 44 amino acids. In contrast the rat GHRH is the 43 amino acid long non-amidated peptide, 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 the signal peptide and proteolytic cleavage at C-terminal region. The amino acid sequence of the precursor comprises 107 or 108 residues.

The GHRH includes five small exons separated by 4 introns and spans over 10 kilobases.

The patent application EP1052286 illustrates the non-specific method to clone the canine GHRH gene from a genomic library but does not disclose the nucleotide sequence of the gene or provides the exon sequences to obtain the full coding sequence.

In farm animals, GHRH is galactopoietic with no alteration in milk composition. By stimulation of GH secretion, the GHRH enhances the immune functions in animals. The 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 abnormalities, in wound healing, bone healing, retardation of the aging process, osteoporosis and anemia (Sohmiya et al. J. Endocrinol. Invest. 2000, 23, 31-36 and Clin. Endocrin. 2001,55, 749-754).

GHH are required to stimulate the production and secretion of GH. GHH, Heterogeneous, GHG, Heterogeneous, GHG, Heterogeneous, or Dysfunctional. The administration of the heterologous GHRH peptide may also be an induce antibody response in the host. The DNA encoding the canine GHRH was unknown until the present description and there was a description of the canine GHRH.

[0011] Citation or disclosure of a document is available as a prior art.

SUMMARY OF THE INVENTION

Applicants' identification of novel canine pre-proGHRH and canine mature GHRH polynucleotide and polypeptide sequences.

IGF-1 signal peptide fused to canine mature GHRH and a c-myc epitope tag, which is a polynucleotide is operatively linked to an enhancer and / or the promoter, and the amino acid sequence shown as amino acids 31-74 of SEQ ID NO: 1 (page 7, paragraph [0049], and pages 31-32, paragraph [0136]).

In a further aspect, the present invention provides a hybrid protein containing equine IGF-1 signal peptide fused to canine proGHRH and a c-myc epitope tag that is operatively linked to an enhancer and / or a promoter, and the amino acid sequence shown as amino acids 20-74 of SEQ ID NO: 1 (page 7, paragraph [0049], and pages 31-32, paragraph [0136]) .

In another aspect, the present invention provides a method for delivery and expression of a canine matrix in a cell, which is the vector of the present invention.

In another aspect, the present invention provides a vector of the present invention and a vector of the present invention.

[0017] In a further aspect, the present invention provides a method of treating anemia, cachexia, obesity or osteoporosis in a vertebrate.

In another aspect, an expression vector of the present invention is the present invention for use in the treatment of anemia, cachexia, obesity or osteoporosis in a vertebrate.

[0019] The present description or a canine pre-proGHRH polypeptide, canine mature GHRH peptide and polynucleotides, which encodes the canine pre-proGHRH or the canine mature GHRH. In an advantageous form, the canine pre-proGHRH polypeptides are derived from the amino acid residues of SEQ ID NO: 1. In another advantageous form, the canine mature GHRH peptide is derived from the amino acid residues of SEQ ID NO: 2.

The description also encompasses an isolated polynucleotide, which is fully complementary, having the sequence of SEQ ID NO: 3 and an isolated polynucleotide, or an antisense beach that is fully complementary, which consists of canine mature GHRH, having the sequence of SEQ ID NO: 4. The polynucleotides can be DNA or RNA molecules.

In one study the description and a vector encoding and expressing the canine pre-proGHRH or the canine GHRH. Such a treatment can be used to treat disease and growth hormone deficiency by gene therapy in vertebrates. The advantage vector is a polynucleotide that encodes a nucleotide base sequence of SEQ ID NO: 3. In another advantageous expression, the expression vector com prises polynucleotide that encodes a canine mature GHRH , wherein the polynucleotide is the nucleotide base sequence of SEQ ID NO: 4. In an advantageous embodiment, the nucleotide base sequence is operatively linked to a promoter.

In a particularly advantageous form, the canine proGHRH is the N-terminus of the canine proGHRH (corresponding to 31-106 AA) or the canine GHRH is a peptide signal sequence. fused to the canine proGH RH, canine proGHRH deleted from the propeptide N-terminus (corresponding to 31-106 AA) or the canine GHRH. Advantageously, the peptide signal sequence is an insulin-like growth factor (IGF) peptide signal sequence. The peptide signal sequence is an advantageous peptide signal sequence, more advantageously, an equine IGF-1 peptide signal sequence.

In another advantageous art, the description encompasses the vector encoding and expressing the GHRH containing a peptide signal sequence, advantageously an IGF-1 peptide signal sequence and a proGHRH. at the C-terms. In yet another advantageous form, a description of a sequence of recombinant peptide signal sequences can be found in a GHRH and a serum album, a linked link through a linker tetrapeptide. The GHRH may be derived from pig or cattle as well as dogs.

Any of the vectors mentioned above may be used to treat disease and growth hormone deficiency by gene therapy in vertebrates. The expression vector is a polynucleotide with the nucleotide base sequence of SEQ ID NO: 3. In another advantageous vector, the expression vector is a polynucleotide that encodes a canine mature GHRH, the polynucleotide comprising the nucleotide base sequence of SEQ ID NO: -4. In an advantageous form, the nucleotide base sequence is an operative linked promoter and optional an enhancer.

In another embodiment, the present invention provides a method for delivering the peptide GHRH to a vertebrate, in particular to a dog, in a canine pre-proGHRH or the canine GHRH to the animal host. In an advantageous, the animal host is a dog. GHRH ora canine pre-proGHRH and a can or pre-proGHRH in a cell or improves the stability of the vector. Advantageously, the delivery is in vivo to an animal, advantageously the vertebrate, more advantageously, the dog. More preferably, the canine pre-proGHRH sequence is composed essentially of SEQ ID NO: 3 of SEQ ID NO: 4.

The description also provides a method for delivering a GHRH to an animal, advantageously a dog, with more injecting a GHRH formulation to the animal. In an advantageous form, the formulation is a vector expressing canine GHRH and is a carrier or vehicle or excipient.

The description and method of treatment of anemia, cachexia, wound healing, bone healing, osteoporosis and obesity in vertebrates. The description also provides a method for stimulating the immune response of a vertebrate.

[0028] It is noted that the term "comprised", "comprised", "as" is "U.S. Patent Law; e.g., they can mean "includes", "included", "including", and the like; and in the form of a statement of the meaning of the term ascribed to them in U.S. Patent law, eg, they do not explicitly recited, but exclude element. As used here, the meaning of explicitly exclude non-canine GHRH sequences.

[0029] These are described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, is given in the following drawings, in which: FIG. 1 depicts the plasmid map and the encoded ORF of pNB179. The nucleotide sequence of the encoded ORF is SEQ ID NO: 49 and SEQ ID NO: 50. 2 depicts the plasmid map and the encoded ORF of pNB209. The nucleotide sequence of the encoded ORF is also SEQ ID NO: 26 of SEQ ID NO: 27. FIG. 3 depicts the plasmid map and the encoded ORF of pNB210. The nucleotide sequence of the encoded ORF is also SEQ ID NO: 28 and SEQ ID NO: 29. 4 depicts the plasmid map and the encoded ORF of pNB211. The nucleotide sequence of the encoded ORF is SEQ ID NO: 30 and the amino acid sequence of the encoded ORF of SEQ ID NO: 31. 5 depicts the plasmid map and the encoded ORF of pNB212. The nucleotide sequence of the encoded ORF is SEQ ID NO: 32 and SEQ ID NO: 33. 6 depicts the plasmid map and the encoded ORF of pNB213. The nucleotide sequence of the encoded ORF is also SEQ ID NO: 34 of SEQ ID NO: 35. FIG. 7 depicts the plasmid map and the encoded ORF of 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. 8 depicts 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. 9 depicts the plasmid map and the encoded ORF of pNB216. The nucleotide sequence of the encoded ORF is SEQ ID NO: 40, and SEQ ID NO: 27. 10 depicts 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. 11 depicts 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. 12 depicts the plasmid map and the encoded ORF of pNB219. The nucleotide sequence of the encoded ORF is SEQ ID NO: 43 and SEQ ID NO: 37. 13 depicts the plasmid map and the encoded ORF of pNB220. The nucleotide sequence of the encoded ORF is also SEQ ID NO: 44 of SEQ ID NO: 39. 13 depicts the plasmid map and the encoded ORF of pNB220. FIG. 14 depicts the plasmid map and the encoded ORF of pNB240. The nucleotide sequence of the encoded ORF is SEQ ID NO: 55 and SEQ ID NO: 56. 15 depicts the plasmid map and the encoded ORF of pNB239. The nucleotide sequence of the encoded ORF is SEQ ID NO: 57 and SEQ ID NO: 58. 16 depicts the plasmid map and the encoded ORF of pNB244. The nucleotide sequence of the encoded ORF is SEQ ID NO: 59 and SEQ ID NO: 60. 17 depicts the plasmid map and the encoded ORF of pNB232. The nucleotide sequence of the encoded ORF is SEQ ID NO: 61 and SEQ ID NO: 62.

FIG. 18 depicts the plasmid map and the encoded ORF of pNB245. The nucleotide sequence of the encoded ORF is SEQ ID NO: 63 and SEQ ID NO: 64. 19 depicts the plasmid map and the encoded ORF of pNB228. The nucleotide sequence of the encoded ORF is SEQ ID NO: 69 and SEQ ID NO: 70. 20 depicts 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. 21 depicts 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 depicts the plasmid map and the encoded ORF of pNB299. The nucleotide sequence of the encoded ORF is SEQ ID NO: 76 and SEQ ID NO: 77. 23 depicts 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

Applicants' identification of novel canine pre-proGHRH and canine mature GHRH polynucleotide and polypeptide sequences. GHRH peptides and coding sequences from different animal species such as mouse, rat, pig and bovine. Using a heterogous GHRH in dog may lead to a less potent stimulation of the host response. The DNA encoding the canine GHRH was unknown until the present description and there was a description of the canine GHRH.

The present description of the present invention provides a pre-proGHRH polypeptide having the following amino acid sequence: H-MPLWVFFLVILTLSSGSHSSPPSLPIRIPRYADAIFTNSYRKVLGQLSARKLLQDIMSRQ QGERNREQGAKVRLGRQVDSLWASQKQMALENILASLLQKRRNSQG-OH (SEQ ID NO: 1). The peptide signal (prepeptide) sequence spans from Met (1) to Ser (20). The signal peptide can also occur after the Ser (19). The amino acid position in the pre-proGHRH sequence. The H-M and G-OH are the amino acid at the terminus of the carboxy terminus of the pre-propeptide are not modified. After cleavage of the preGHRH peptide, the proGHRH peptide is cleaved after the Arg (30) and after the Leu (74) to the lead GHRH peptide.

A variant of the canine pre-proGHRH polypeptide is a substitution of amino acid Pro in position 2 by the amino acid Leu.

[0034] The present description is also a canine mature GHRH peptide having the following amino acid sequence: R1-YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGERNREQGAKVRL-R2 (SEQ ID NO: 2) and R- is hydrogen or Met-Met and R2 is OH or NH2. The Met-Met or the H-Y is the term of the methionine or the tyrosine are not modified. The L-OH or L-NH2 at the carboxy terminus of the mature peptide means that the leucine amino acid is either unmodified or amidated.

In another art the description includes a canine mature GHRH analogue with improved stability. (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). Parentheses means the amino acid position in the mature GHRH sequence. These amino acid substitutions can be introduced in the canine pre-proGHRH polypeptide. It is routine experimentation forone of skill in the art of amino acid substitutions. For example, but not by limitation, site-directed mutagenesis of the canine mature GHRH polynucleotide can be carried out with a mutant canine mature GHRH peptide with one or more of the above-listed amino acid substitutions. The gene can also be synthetized by methods known in the art.

The terms " protein ", " peptide ", " polypeptide " and " polypeptide fragment " are used interchangeably for amino acid residues of any length. The amino acids or amino acid analogues of the polymer may be used. An acidic polymer that has been modified; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or other manipulation or modification.

The present description encompasses an isolated polynucleotide encoding the canine pre-proGHRH having the sequence and fragments:

5'-ATGCCACTCTGGGTGTTCTTCCTGGTGATCCTCACCCTCAGCAGTGGCTCCCACTC TTCCCCGCCATCCCTGCCCATCAGAATCCCTCGGTATGCAGACGCCATCTTCACC AACAGCTACCGGAAGGTGCTGGGCCAGCTGTCCGCCCGCAAGCTCCTGCAGGAC ATCATGAGCCGGCAGCAGGGAGAGAGAAACCGGGAGCAAGGAGCAAAGGTACG ACTCGGCCGTCAGGIGGACAGTCTGTGGGCAAGCCAAAAGCAGATGGCATTGGA GAACATCCTGGCATCCCTGTTACAGAAACGCAGGAACTCCCAAGGATGA-35 (SEQ ID NO: 3).

The description also provides a variant of the polynucleotide encoding the canine pre-proGHRH substitution of the nucleotides C and A in position 5 and 6 by T and G, respectively.

The present description also includes an isolated polynucleotide encoding the canine mature GHRH having the sequence: 5 TATGCAGACGCCATCTTCACCAACAGCTACCGGAAGGTGCTGGGCCAGCTGTCCG CCCGCAAGCTCCTGCAGGACATCATGAGCCGGCAGCAGGGAGAGAGAAACCGG GAGCAAGGAGCAAAGGTACGACTC-3 '(SEQ ID NO: 4).

A "polynucleotide" is a polymeric form of any length, which contains deoxyribonuelcotides, ribonucleotides, and analogs in any combination. Polynucleotides may have three-dimensional structure, and may perform any function, known or unknown. The term "polynucleotide" includes double-, single-stranded, and triple-helical molecules. Unless otherwise specified or required, any description of the description is a polynucleotide encompasses both the double stranded form of the DNA, RNA or hybrid molecule.

The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide may be modified nucleotides and nucleotide analogs, uracyl, and other linking groups such as fluororibose and thiolate, and nucleotide branches. The sequence of nucleotides may be further modified by polymerization. Other types of polynucleotides or solid support, other than polynucleotides or solid support.

An "isolated" polynucleotide or polypeptide is one that is free of the materials. At least 50%, advantageously at least 70%, more advantageously at least 80%, and even more at least 90% free of these materials.

The description further includes a complementary strand to the GHRH polynucleotide.

The complementary strand can be polymeric and of any length, and may contain deoxyribonucleotides, ribonucleotides, and analogs in any combination.

Hybridization reactions can be performed under different conditions "stringency." Conditions for a hybridization reaction are well known. See for example, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al. 1989). Examples of relevant conditions include: incubation temperatures of 25 ° C, 37 ° C, 50 ° C, and 68 ° C; buffer concentrations of 10 x SSC, 6 x SSC, 1 x SSC, 0.1 x SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer) and their equivalent using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutes to 24 hours; 1,2 or more washing steps; wash incubation times of 1.2, or 15 minutes; and wash solutions of G x SSC, 1 x SSC, 0.1 x SSC, or deionized water.

The description further encompasses polynucleotides encoding functionally equivalent variants and derivatives of GHRH polypeptides and functionally equivalent fragments. These functionally equivalent variants, derivatives, and fragments display the ability to retain canine GHRH activity. For instance, changes in a DNA sequence that do not change the amino acid residues, and a substitution of amino acid residues; acid analogs are those which are not affected by the encoded polypeptide. Conservative amino acid substitutions are glycine / alanine; valine / isoleucine / leucine; asparagine / glutamine; aspartic acid / glutamic acid; serine / threonine / me-thionine; lysine / arginine; and phenylalanine / tyrosine / tryptophan.

At least 90%, at least 90%, 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% or at least 99% homology or identity to SEQ ID NO: 1.

In another embodiment the description includes a canine mature 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 SEQ ID NO: 2 .

At least 88%, at least 88%, at least 88.5%, at least 89%, at least 88%, at least 89%, at least 88%, at least 89% at least 90%, 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 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% or at least 99%, at least 99.5% homology or identity to SEQ ID NO: 3. The description encompasses also a polynucleotide encoding a canine pre-proGHRH analogue. At least 89%, at least 89%, at least 89.5%, at least 90%, at least 89.5%, at least 90%, at least 89.5%, at least 90% at least 92%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, 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%, or at least 99% or at least 99.5% homology or identity to SEQ ID NO: 1.

At least 92%, at least 92%, at least 92.5%, at least 93%, at least 92%, at least 93%, at least 92.5%, at least 93%, at least 92.5%, at least 93%, at least 92.5%, at least 93%, at least 92.5% 93.5%, at least 94%, at least 94.5%, at least 95%, at least 96.5%, at least 97%, at least 97.5%, at least 98% at least 98.5%, at least 99% or at least 99.5% homology or identity to SEQ ID NO: 4. The description encompasses also a polynucleotide encoding with a canine mature GHRH analogue. In an advantageous form, the canine mature GHRH analogue 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 description, sequencing or homology is determined by comparing the sequences when minimizing sequence gaps. A number of mathematical algorithms. A nonlimiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin &amp; Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268, modified as in Karlin &amp; Altschul, Proc. Natl. Acad. Sci. USA 1993; 90: 5873-5877.

Another example of a mathematical algorithm for comparing the sequences of the algorithm of Myers &amp; Miller, CABIOS 1988; 4: 11-17. Such an algorithm is included in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, the PAM120 weight loss table can be used. Yet another useful algorithm for the identification of the local sequence similarity and alignment is the FASTA algorithm as described in Pearson &amp; Lipman, Proc. Natl. Acad. Sci. USA 1988; 85: 2444-2448.

Advantageous for use according to the present description is the WU-BLAST (Washington University BLAST) version 2.0 software. WU-BLAST version 2.0 for several UNIX platforms can be downloaded from ftp: // blast. wustl. edu / blast / Executables. Version 1.4, which is on the public domain NCBI-BLAST version 1.4 (Altschul &amp; Gish, 1996, Local alignment statistics, Doolittle ed., Methods in Enzymology 266: 460-480; Altschul et al., Journal of Molecular Biology 1990; 215: 403-410; Gish &amp; States, 1993; Nature Genetics 3: 266-272; Karlin &amp; Altschul, 1993; Proc. Natl. Acad. Sci. USA 90: 5873-5877.

In general, comparison of amino acid sequences is accomplished by aligning an amino acid sequence of a polypeptide of the unknown structure. Amino acids in the sequences are then grouped together. This is a method for detecting and analyzing amino acid insertions and deletions. Homology between amino acid sequences can be determined by commercially available algorithms (see also the description of homology above). BLAST, Gapped BLAST, BLASTN, BLASTP, and PSI-BLAST provided by the National Center for Biotechnology Information. These programs are widely used in the art of two amino acid sequences.

In all search programs are the gapped alignment routines are integral to the database search itself. Gapping can be turned off if desired. The default penalty (Q) for a gap of length is Q = 9 for BLASTTP, and Q = 10 for BLASTN, but may be changed to any integer. R = 2 for proteins and BLASTP, and R = 10 for BLASTN, but may be changed to any integer. Any combination of values for Q and R can be used in order to minimize sequences of gaps. The default amino acid comparison matrix is BLOSUM62, but other amino acid comparison matrix such as PAM can be utilized.

Alternatively or additionally, the term "homology" or "identity" for instance, with respect to a nucleotide or amino acid sequence, may be used. (Nref - Nd / f) * 100 / Nref, ndf is the total number of non-identical residues in one of the two sequences. of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (Nref = 8; Nd / i = 2).

Alternatively or additionally, "homology" or "identity" with respect to the sequences of the nucleotides or amino acids in the shorter of the two sequences is alignment of (Wilbur &amp; Lipman, Proc Natl Acad Sci USA 1983; 80: 726), for instance, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and computer-assisted analysis (eg Intelligenetics ™ Suite, Intelligenetics Inc. CA). When RNA sequences are, the DNA sequence is considered to be uracil (U) in the RNA sequence. Thus, RNA sequences are within the scope of DNA sequences, by thymidine (T) in the DNA sequence being considered to be uracil (U) in RNA sequences.

And, without undue experimentation, the skilled artisan can consult with many other programs or references for determining percent homology.

The description further encompasses the canine GHRH polynucleotides contained in a vector molecule or an expression vector and a promoter element and optional to an enhancer.

The description further encompasses the canine GHRH polynucleotides contained in a vector molecule or an expression vector, a canine proGHRH deleted from the propeptide N-terminus (corresponding to 31-106 AA) or the canine GHRH. Advantageously, the peptide signal sequence is an insulin-like growth factor (IGF) peptide signal sequence. The peptide signal sequence is an advantageous peptide signal sequence, more advantageously, an equine IGF-1 peptide signal sequence. N-terminus of the propeptide (corresponding to 31-106 AA) or the canine GHRH nucleotide sequence fused to the peptide signal sequencing by removing cryptic splice sites , eg, by adapting the codon usage by introducing a Kozak consensus sequence before the start codon to improve expression.

In an advantageous embodiment, the promoter of the cytomegalovirus (CMV) is an immediate early gene. In another advantageous, the promoter and / or enhancer elements are oxygen-inducible. Examples of oxygen-inducible promoters and / or enhancers that can be used include, but are not limited to, early growth response-1 (Egrl) promoter (eg, Park et al. J Clin Invest 2002 Aug; 110 (3): 403-1), hypoxia-inducible factor (HIF) inducible enhancers (e.g., Cuevas et al., Cancer Res. Oct. 15, 2003; 63 (20): 6877-84) and Mn superoxide dismutase (Mn-SOD) promoter (eg, Gao et al., Gene. 1996 Oct 17; 176 (1-2): 269-70).

Other GHRH DNA sequences are isogeneously specific for, e.g., muscle, endothelial cell, liver, or somatic cell or stem cell. each animal species. For example, if the GHRH is expressed in a canine muscle cell, the enhancers and / or promoters of the canine GHRH. Examples of muscle-specific promoters and enhancers are those known in the art (eg, Li et al., Gene Ther. 1999 Dec; 6 (12): 2005-11; Li et al. , Nat Biotechnol. 1999 Mar; 17 (3): 241-5 and Loirat et al., Virology 1999 Jul 20; 260 (1): 74-83).

Promoters and enhancers that are employed in the present invention include, but are not limited to, LTR or Rous sarcoma virus, TKofHSV-1, early or late promoter of SV40, adenovirus major late (MLP), phosphoglycerate kinase, metallothionein , a-1 antitrypsin, albumin, collagenase, elastase I, β-actin, β-globin, γ-globin, α-fetoprotein, muscle creatin kinase.

A "vector" refers to a recombinant DNA or RNA plasmid or virus that comprises a heterologous polynucleotide to a target cell, in vitro or in vivo. The heterologous polynucleotide may be a sequence of interest for an expression cassette. As used in, the vector is not capable of replication in the ultimate target cell or subject. The term includes cloning vectors also included in viral vectors.

[0066] The term "recombinant" means a polynucleotide semisynthetic, or does not occur in nature.

"Heterologous" means a genetically distinct entity. For example, a polynucleotide may be inserted by a genetic engineering technique into a plasmid or a heterologous polynucleotide. The promoter is a heterologous promoter.

Additional transcription units, such as promoters, ribosome binding sites, polyadenylation sites, additional transcription units, and other transcription units, promoter, sequences that permit cloning, expression, homologous recombination, and transformation of a host cell.

The present description encompasses a vector expressing pre-proGHRH, the mature GHRH, the proGHRH or variant or analogues or fragments. For the mature GHRH or the proGHRH, the nucleotide sequence encoding the peptide is preceded by a nucleotide sequence in a cell encoding a peptide signal. A peptide signal from a secreted protein, e.g. the peptide from the tissue plasminogen activator protein (tPA), in particular the human tPA (S. Friezner Degen et al. J. Biol. Chem. 1996, 261, 692-6985; R. Rickies et al., J. Biol. Chem. 1988 , 263, 1563-1569, D. Berg, et al. Biochem Biophys Res Commun., 1991, 179, 1289-1296, or the signal peptide from the Insulin-like growth factor 1 (IGF1), in particular the equine IGF1 (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. Genome 2000, 11 (10), 877-882), the porcine IGF1 (M. Muller et al., Nucleic Acids Res. 1990, 18 (2), 364), the chicken IGF1 (Y. Kajimoto et al. Mol. Endocrinol. 1989, 3 (12), 1907-1913), the turkey IGF1 (Genbank accession number AF074980). The signal peptide from IGF1 may be natural or optimized by cryptic splice sites and / or by adapting the codon usage. For the mature GHRH Gly and Arg can be added to the C-terminus of the peptide in the coding polynucleotide sequence in order to obtain amidation.

[0070] Elements for the expression of canine GHRH are advantageously present in an inventive vector. In at least a continent, this composition, or a termination codon and a promoter, and a polyadenylation sequence for certain vectors such as plasmids and viral vectors. than poxviruses. When the polynucleotide encodes a polyprotein fragment, e.g. canine GHRH, advantageously, in the vector, an ATG is placed at 3 '. Other elements for controlling expression may be present, such as intrinsic and signal sequences.

Methods for making and / or administering a vector or recombinant expression of the genes of the description or in vivo or in vitro method disclosed in: or in documents cited in: US 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; U.S. Patent Application Serial No. 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. Natl. Acad. Sci. USA 1996; 93: 11313-11318; Ballay et al., EMBO J. 1993; 4: 3861-65; Feigner et al., J. Biol. Chem. 1994; 269: 2550-2561; Frolov et al., Proc. Natl. 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., Diabetology 1998; 41: 736-739; Kitson et al., J. Virol. 1991; 65: 3068-3075; McClements et al., Proc. Natl. Acad. Sci. USA 1996; 93: 11414-11420; Moss, Proc. Natl. Acad. Sci. USA 1996; 93: 11341-11348; Paoletti, Proc. Natl. 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. Natl. Acad. Sci. USA 1996; 93: 11334-11340; Robinson et al., Sem. Immunol. 1997; 9: 271; and Roizman, Proc. Natl. Acad. Sci. USA 1996; 93: 11307-11312. Such as a poxvirus (eg, vaccinia virus, avipox virus, canarypox virus, fowlpox virus, raccoonpox virus, swinepox virus, etc.), adenovirus (eg, human adenovirus, canine adenovirus), herpesvirus (eg canine herpesvirus), baculovirus, retrovirus, etc .; or the vector can be a plasmid. Non-canine mature GHRH peptides, non-canine pre-proGHRH peptides \ t , non-canine preGHRH peptides, non-canine proGHRH peptides or fragments, cytokines, etc. the composition of the description.

The present disclosure also relates to vectors, such as expression vectors, e.g., therapeutic compositions. Expression vectors, such as in vivo expression vectors, such as in vivo expression vectors; Advantageously, the vector contains and expresses a polynucleotide that includes, or contains, a coding region. Thus, a GHRH or a fragment of a cannabis is a GHRH or a fragment thereof .

[0073] According to another embodiment of the present invention, the vector or vectors have an expression or an expression of a protein. of the polynucleotide (s). At least two vectors include, or, in particular, expressing, advantageously in vivo, or in a suitable host cell, polynucleotides from different canine GHRH isolates encoding the same proteins and / or different proteins, but advantageously for the same proteins. Preparations containing one or more vectors containing, in particular, polynucleotides encoding, and advantageously expressing, advantageously in vivo, canine GHRH peptide, fusion protein or an epitope. GHRH, eg, GHRH, GHRH, cows, goats, humans, \ t mice, monkeys, pigs, rat and sheep, in addition to dogs.

Expression vector is, according to one of the description, the expression vector. The expression vector is an adenovirus vector. Advantageously, the adenovirus is a human Ad5 vector, an E3-deleted adenovirus.

In one particular embodiment, the viral vector is a poxvirus, e.g. vaccinia virus or an attenuated vaccinia virus (for instance, MVA, a modified ankara strain); this Stickl & Hochstein-Mintzel, Munch Med. Wschr. 1971, 113, 149-1153; Sutter et al., Proc. Natl. Acad. Sci. USA, 1992, 89,10847-10851; available as ATCC VR-1508; or NYVAC, U.S. Patent No. 5,494,807, for instance Of theVVV with additional ORFs, as well as the insertion of heterologous coding nucleic acid molecules into sites an avipox virus or an attenuated avipox virus (eg, canarypox, fowlpox, dovepox, pigeonpox, quailpox, ALVAC orTROVAC; this, eg, US Patent) No. 5,505,941,5,494,807), swinepox, raccoonpox, camelpox, or myxomat osis virus.

According to another example, the poxvirus vector is a canarypox virus or a fowlpox virus vector, advantageously an attenuated canarypox virus or fowlpox virus. VR-111 is available at the ATCC under access number VR-111. Attenuated canarypox viruses are described in U.S. No. 5,756,103 (ALVAC) and W001 / 05934. Numerous fowlpox virus vaccination strains are also available, e.g. the DIFTOSEC CT strain marketed by MERIAL and the NOBILIS VARIOLE vaccine marketed by INTERVET; and, reference is also made to U.S. Patent No. 5,766,599 which pertains to the atenuated fowlpox strain TROVAC.

U.S. Pat. No. 3,973,196 to U.S. Patent Application Publication No. U.S. Pat. Patents Well. 4,769,330, 4,722,848, 4,603,112, 5,110,587, 5,494,807, and 5,762,938 inter alia] as fowlpox, mention is made of U.S. Patents Well. 5,174,993, 5,505,941 and US-5,766,599 inter alia] as canarypox mentionis made of U.S. U.S. Patent No. 5,756,103 inter alia. 5,382,425 inter alia] and, as to raccoonpox, mention is made of WO00 / 03030 inter alia.

When the expression vector is vaccinia virus, insertion site or the polynucleotide or polynucleotide to be expressed is the site of the haemagglutinin (HA) gene or insertion site, the region encoding the inclusion body of the A type; it is also the case for vaccinia virus. In the case of canarypox, advantageously the insertion site or sites are ORF (s) C3, C5 and / or C6; it is also referred to as a canarypox virus. In the case of fowlpox, advantageously the insertion site or sites are ORFs F7 and / or F8; See also the documentary reference virus. The insertion site or sites for MVA virus area, including various publications, including Carroll 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; The genome of the complete MVA is described in Antoine G., Virology, 1998, 244, 365-396, which provides the skilled artisan to use other insertion sites or other promoters.

Advantageously, the polynucleotide to be expressed is the specific poxvirus promoter, eg, the vaccinia promoter 7.5 kDa (Cochran et al., J. Virology, 1985, 54, 30-35), the vaccinia promoter 13L (Riviere et al., J. Virology, 1992, 66, 3424-3434), the vaccinia promoter HA (Shida, Virology, 1986, 150, 451-457), the cowpox promoter ATI (Funahashi et al., J. Gen. Virol., 69, 35-47 (1988), 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., 63, 3829-3836 (1989), inter alia.

In particular, the viral vector is an adenovirus, such as a human adenovirus (HAV) or a canine adenovirus (CAV).

In one of the viral vector is the human adenovirus, in particular the serotype 5 adenovirus, rendered incompetent by the deletion in the E1 region of the viral genome, in particular from about nucleotide 3510 by reference to published in Genbank under the accession number M73260 and in the referenced publication J. Chroboczek et al Virol. 1992, 186, 280-285. The adenovirus is propagated in E1-expressing 293 (F. Graham et al. Gen. Virol. 1977, 36, 59-72) or PER cells, in particular PER.C6 (F. Falloux et al. 9, 1909-1917). The deletion in the E1 region (i.e., J. Shriver et al., Et al., Et al., Supra). 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, pp. 109-128; al. Proc. Natl. Acad. Sci. 1997, 94, 2587-2592; US6,133,028; US6,692,956; S. Tripathy et al. Proc. Natl. 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. Res. 1983, 11,6003-6020; C. Chavier et al J. Virol., 1996, 70, 4805-4810). The insertion sites can be the E1 and / or E3 loci (region). Advantageously, a cytomegalovirus immediate-early gene promoter (CMV-IE promoter), in which the expression vector is an adenovirus; particular the enhancer / 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 from the pCI vector from Promega Corp. The CMV-IE promoter advantageously of murine or human origin. The promoter of the elongation factor 1a can also be used. In one particular aspect, the promoter is regulated by hypoxia, e.g. the promoter HRE described in K. Boast et al. Human Gene Therapy 1999, 13, 2197-2208), can be used. The muscle specific promoter can also be used (X. Li et al. Nat. Biotechnol. 1999, 17, 241-245). Strong promoters are also discussed in relation to plasmid vectors. In one study, a splicing sequence can be located downstream of the enhancer / promoter region. For example, the intron 1 is isolated from the CMV-IE gene (R. Stenberg et al. J. Virol. 1984, 49, 190), the intron isolated from the rabbit or human β-globin gene, in particular the intron. b-globin gene, the intron isolated from the immunoglobulin gene, a splicing sequence from the SV40 early gene or the human β-globin donor sequence fused to the mouse immunoglobulin acceptor sequence (from about nucleotide 890 to about nucleotide 1022 in Genbank under the accession number CVU47120). A poly (A) sequence and terminator sequence can be inserted into the polynucleotide to be expressed, e.g. a bovine growth hormone gene, in particular from about nucleotide 2339 to about nucleotide 2550 in Genbank under the accession number BOVGHRH, a rabbit β-globin gene or SV40 late gene polyadenylation signal.

CAV-2 (e.g., Fischer et al. Vaccine, 2002, 20, 3485-3497; US Patent No. 5,529,780; U.S. Patent No. 5,529,780; U.S. Patent No. 5,529,780; 5,688,920; PCT Application No. WO95 / 14102). For CAV, the insertion sites can be in the region E3 region and the right ITR region (U.S. Patent No. 6,090,393; U.S. Patent No. 6,156,567). A cytomegalovirus immediate-early gene promoter (CMV-IE promoter) or a promoter already described for a human adenovirus vector. A poly (A) sequence and terminator sequence can be inserted into the polynucleotide to be expressed, e.g. a bovine growth hormone gene or a rabbit β-globin gene polyadenylation signal.

A viral vector is a herpesvirus such as canine herpesvirus (CHV) or feline herpesvirus (FHV). For CHV, the insertion sites may be in particular in the thymidine kinase gene, in ORF3, or in the UL43 ORF (U.S. Patent No. 6,159,477). In one aspect, the polynucleotide is expressed as a promoter of functional eukaryotic cells, preferably CMV-IE promoter (murine or human). In one particular aspect, the promoter is regulated by hypoxia, e.g. the promoter HRE described in K. Boast et al. Human Gene Therapy 1999, 13, 2197-2208), can be used. A poly (A) sequence and terminator sequence can be inserted into the polynucleotide to be expressed, e.g. bovine growth hormone or a rabbit β-globin gene polyadenylation signal.

Plasmid vector or a DNA plasmid vector, in particular an in vivo expression vector. In a specific, non-limiting example, the pVR1020 or 1012 plasmid (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, e. G., US Patent Nos. 5,846,946 and 6,451,769) for use in a polynucleotide sequence. The pVR1020 plasmid is derived from the pVR1012 and contains the human tPA signal sequence. In one study the amino acid M (1) is the amino acid S (23) in Genbank under the accession number HUMTPA14. In another specific, non-limiting example, the plasmid is utilized as a vector for the insertion of a signal peptide sequence of equine IGF1 from amino acid M (24) to amino acid A (48) in Genbank under the accession number U28070. Additional information about DNA plasmids which may be found in the practice is found, for example, in U.S. Patent 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 DNA transcription unit a polynucleotide according to the invention; a circular plasmid, as well as a linear form, is intended to be within the scope of the description.

GHRH deleted of the propeptide N-terminus of GHRH, each of which is a plasmid nucleotide encoding GHRH, a canine proGHRH, the canine proGHRH fused with a heterologous peptide sequence. (corresponding to 31-106 AA), variant, analog or fragment, operably linked to a promoter or promoter. In general, it is advantageous to employ a strong promoterfunctional in eukaryotic cells. The preferred strong promoter is the immediate early cytomegalovirus promoter (CMV-IE) of human or murine origin. The CMV-IE promoter can be the actual promoter part. EP-A-260 148, EP-A-323 597, U.S. Pat. Patents Well. 5,168,062, 5,385,839, and 4,968,615, as well as to PCT Application No. WO87 / 03905. The CMV-IE promoter is advantageously a human CMV-IE (Boshart M. et al. Cell., 1985, 41, 521-530) or murine CMV-IE.

In general, the promoter has a viral or a cellular origin. The strong viral promoter other than CMV-IE is a viral or LTR promoter of the Rous sarcoma virus. The strong cellular promoter that may be useful is the invention of the cytoskeleton, such as e.g. 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 sub fragments of these promoters, i.e., portions of these promoters, are included within the present description, e.g. truncated CMV-IE promoter according to PCT Application N ° W098 / 00166 or U.S. Patent No. 6,156,567 can be used in the practice of the invention. The promoter is in the practice of the invention as a promoter, which is a promoter and promoter. the derivative or sub fragment is derived, eg, the activity of the truncated CMV-IE promoter of US Patent No. 6,156,567 to the activity of full-length CMV-IE promoters. Thus, the CMV-IE promoter in the practice of the full-length promoter and / or enhancer portion of thefull-length promoter, as well as derivatives and subfragments.

Preferably, the plasmids are other expression control elements. It is particularly advantageous to incorporate a stabilizing sequence (s), an intron sequence (s), a first intron of the hCMV-IE (PCT Application No. WO89 / 01036), the intron II of the rabbit β-globin gene ( van Ooyen et al., Science, 1979, 206, 337-344).

The polyadenylation signal (polyA) for plasmids and viral vectors other than poxviruses is a poly (A) signal of the bovine growth hormone (bGH) gene (U.S. Patent No. 5,122,458) , or the poly (A) signal of the SV40 virus.

N-terminus of IHR1 and propeptide GHRH deleted of the propeptide (corresponding to 31-106 AA). The expression vector of the IGF1 and the mature GHRH (corresponding to 31-74 AA). In another advantageousad, the description encompasses the vector encoding and expressing the GHRH containing a peptide signal sequence, advantageously an IGF-1 peptide signal sequence and a proGHRH deleted -term. In yet another advantageous form, a description of a sequence of recombinant peptide signal sequences can be found in a GHRH and a serum album, a linked link through a linker tetrapeptide. The GHRH may be derived from pig or cattle as well as dogs.

[0092] According to another aspect of the present invention, the expression vectors are used in an in vitro expression system. (C). \ T (c). \ T (c). \ T (c). \ T such as affinity, ion exchange or gel filtration-type chromatography methods.

[0093] Host cells that can be used in the present description include, but are not limited to, muscle cells, keratinocytes, myoblasts, Chinese Hamster ovary cells (CHO), vero cells, BHK21, sf9 cells, and the like. GHRH depends on the host cell. For example, the vector encoding canine GHRH can be transformed into the myoblasts, and the transformed myoblasts can be transplanted to the animal. As a result, myoblasts are genetically engineered to express recombinant canine blood. In another example, keratinocytes can also be transformed with GHRH and transplanted into the animal, resulting in secretion of canine GHRH into circulation.

"Host cell" denotes a prokaryotic or eukaryotic cell that has been genetically altered by an exogenous polynucleotide, such as a recombinant plasmid or vector. When it comes to genetically altered cells, the term refers to the originally altered cell and to the progeny.

Polynucleotides of a desired sequence can be inserted into a suitable vector for cloning or expression. Polynucleotides can be introduced in the art. The vectors containing the polynucleotides of interest can be found in the following: endocytosis, transfection, fitting, electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE- dextran, or other substances; microprojectile bombardment; Lipofectin; and infection (retroviral vector). The choice of introducing vectors or polynucleotides will depend on the host cell.

In an advantageous form, the description provides a therapeutically effective amount of a canine GHRH in a target cell. Determination of the therapeutically effective amount of routine experimentation for one of the ordinary skill in the art. In one of the formulations is an expression vector of a polynucleotide that expresses canine GHRH and aorgan or acceptable carrier, vehicle or excipient. In an advantageous form, the vehicle or excipient facilitates transfection and / or improves preservation of the vector or protein.

[0097] The carriers or vehicles are excipients well known in the art. A 0.9% NaCl (e.g., saline) solution or a phosphate buffer. Otherer or veterinarily acceptable carriers or excipients that may be used include poly- (L-glutamate) or polyvinylpyrrolidone. A carrier or vehicle or excipient may be used as an in-carcinogen or in a vehicle. \ T improve preservation of the vector (or protein). Doses and Dosage are in General, without any undue experimentation.

The cationic lipids containing a quaternary ammonium salt which are advantageously for the following formula:

in which is a saturated or unsaturated straight-chain aliphatic radical having 12 to 18 carbon atoms, R2 is an aliphatic radical containing 2 or 3 carbon atoms and X is an amine or hydroxyl group, e.g. the DMRIE. In another cationic lipid can be associated with a neutral lipid, e.g. the DOPE.

Among these cationic lipids, preference is given to DMRIE (N- (2-hydroxyethyl) -N, N-dimethyl-2,3-bis (tetrade-cyloxy) -1-propane ammonium; WO96 / 34109), advantageously associated with a neutral lipid, advantageously DOPE (dioleoyl-phosphatidyl-ethanol; Behr JP, 1994, Bioconjugate Chemistry, 5, 382-389) to form DMRIE-DOPE.

Advantageously, the plasmid mixture is an extemporaneously and advantageously contemporaneously with administration of the preparation; for instance, the plasmid-adjuvant mixture is formed, advantageously so as to give sufficient time for administration of the mixture to form a complex, e.g. between about 10 and about 60 minutes prior to administration, such as approximately 30 minutes prior to administration.

When DOPE is present, the DMRIE: DOPE molar ratio is advantageously about 95: about 5 to about 5: about 95, more advantageously about 1: about 1, e.g., 1: 1.

The DMRIE or DMRIE-DOPE adjuvant: plasmid weight ratio 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 about 1: about 2, eg, 1: 1 and 1: 2.

In a specific, the pharmaceutical composition is directly administered in vivo, and the encoded product is expressed in the vector in the host. The methods of in vivo delivery a vector encoding GHRH (e.g., U.S. Patent No. 6,423,693; patent publications EP1052286, EP1205551, U.S. Patent Publication 20040057941, WO 9905300 and Draghia-Akli et al., Mol Ther. 2002 Dec; ): 830-6) can be modified to the canine GHRH of the present description to a dog. The in vivo delivery of a canine GHRH described in the art of the above-mentioned reference.

Advantageously, the pharmaceutical and / or therapeutic compositions and / or formulations of the present invention; and, without undue experimentation.

And / or pharmaceutical formulations, and, in particular, about 1 μg to about 2000 μg, advantageously about 50 μg to about. 1000 µg and more preferably from about 100 µg to about 800 µg of plasmid expressing GHRH. Plasmid vector is an electroporation of about 0.1 µg and 1 mg, advantageously between about 2 µg and 50 µg. The dose is about 0.1 and about 2 ml, preferably between about 0.2 and about 1 ml. These doses and doses are suitable for the treatment of canines and other target species such as equines and felines.

10 to 10 to about 1010 and more advantageously from about 106 to about 109 viral particles of recombinant adenovirus expressing GHRH. A dose of about 102 pfu and about 109 pfu. The recombinant expressing GHRH is the recombinant expression of about 106 to 108 pfu of poxvirus or herpesvirus.

Dosage of compositions for target species that are mammals, eg, non-poxvirus-viral-vector-based compositions, is generally between 0.1 and about 2.0 ml, preferably between about 0.1 to about 1.0 ml, and more about 0.5 ml to about 1.0 ml.

[0108] It should be understood by one of the skill in the art that the present invention is not limited. Without an undue experimentation.

[0109] The present description contemplates at least one administration to the animal. The female may be male, female, pregnant female and newborn. Intramuscular (IM), intradermal (ID) or subcutaneous (SC) injection or intranasal or oral administration. The bioassay or Vitajet apparatus (Bioject, Oregon, USA)). Another approach to administer electroporation (eg, S. Tollefsen et al. Vaccine, 2002, 20, 3370-3378; S. Tollefsen et al., J. Immunol., 2003, 57, 229-238) S. Babiuk et al., Vaccine, 2002, 20, 3399-3408; PCT Application No. WO99 / 01158). In another, the plasmid is delivered to the animal by a gene gun or gold particle bombardment. The animal is a vertebrate. In a more advantageous, the vertebrate is a dog.

Canine GHRH, canine GHRH, canine pre-proGHRH, canine proGHRH, variant, or a pharmaceutical composition for the treatment of a disease condition. For example, the secretion of GHH is stimulated by the GHRH. However, cachexia, in particular, cachexia resulting in cancer, healing, bone healing, osteoporosis, obesity, and the like in a vertebrate. The vector expressing canine GHRH of the present description is a 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 growth hormone production abnormalities, osteoporosis and in anemia (Sohmiya et al J. Endocrinol. Invest. 2000, 23, 31-36 and Clin. Endocrin. 2001, 55, 749-754) .

Intramuscular delivery of a plasmid encoding porcine GHRH has been shown to stimulate growth hormone and IGF-I release in dogs to treat anemia and cachexia (see, e.g., Draghia-Akli et al., Mol Ther. 2002 Dec. 6): 830-6). Chronic administration of GHRH has been shown to promote bone healing in dogs (e.g., Dubreuil et al., Can J Vet Res. Jan. Jan. 60 (1): 7-13). It would be advantageous to administer the therapeutic canine GHRH vector of the present description of the polyethylene rod surgical implant of Dubreuil et al. (Can J Vet Res. 1996 Jan; 60 (1): 7-13) to promote bone growth in an animal. Similarly, the GHRH has been shown to promote wound healing (see, e.g., Garrel et al., Surg Res. 1991 Oct; 51 (4): 297-302). Again, it would be advantageous to treat the GHRH vector of the present description of the slow-release pellet implant implant of Garrel et al. (Surg Res. 1991 Oct; 51 (4): 297-302) to promote wound healing in an animal.

The description also provides a method for stimulating the immune response of a vertebrate. Cat, cow, dog, fish, goat, horse, human, mouse, monkey, pig, rat or sheep. In a more advantageous, the vertebrate is a dog.

Because lymphocytes express GH-IGF-I, GHRH and their respective receptors, administration of GHRH has been hypothesized to enhance immune cell function (i.e., Khorram et al., J Clin Endocrinol Metab. 1997).

Nov; 82 (11): 3590-6). In humans, administration of GHRH analogue to healthy individuals, in a profound immune-enhancing effect (see, eg, Khorram et al., J Clin Endocrinol Metab. Nov. 1997; ): 3590-6). Overpexression of GHRH in transgenic mice led to significant stimulation of some parameters of immune function (see, e.g., Dialynas et al., J Clin Endocrinol Metab. 1997 Nov; 82 (11): 3590-6). In another mouse study, GHRH played a crucial role in the development of the experimental autoimmune encephalomyelitis and may provide the basis for a therapeutic approach (this, eg, Ikushima et al., J Immunol. 6): 2769-72). GHRH enhances immune cell function, administration of the therapeutic canine GHRH vector of the present description would be advantageous for stimulation of an immune response in a vertebrate.

[0114] The invention will now be further described by the following non-limiting examples.

EXAMPLES

Example 1.

[0115] Hypothalamus was taken from a liquid nitrogen. Total RNAs were derived from the QIAGEN (Rneasy Mini Protocol for Isolation of Total RNA Catalog ref. 74104). The cells from the hypothalamus were dissociated with a pottery of 600 μΙ of denaturing solution from the kit. This solution contains guanidinium isothiocyanate and beta-mercaptoethanol. The tissue homogenate was centrifuged for 5 minutes at 14,000 RPM (rotations per minute) to remove debris. 600μΙ of a 70% ethanol solution was added and the mixture was loaded onto a Rneasy colomn and centrifuged for 15 seconds at 10,000 RPM. The column was rinsed two times with the RW1 buffer provided with the kit. The RNA was eluted with 50 μΙ RNAse-free buffer after centrifugation 1 minute at 14,000 RPM.

The cDNAs were synthesized in a 20 µl reaction mixture containing 5 mM MgCl2.20 mM Tris HCl pH 8.3.100mM KCl, 1 mM DTT, 1 mM each dNTP, 20 units RNAse inhibitor, 50 units Moloney murine leukemia virus reverse transcriptase, 2.5 μΜ random hexanucleotide primers and 2 μΙ of total hypothalamic RNA. The reverse transcription was 23 ° C for 5 minutes, 42 ° C for 20 minutes, 99 ° C for 5 minutes and 10 ° C for 5 minutes.

The cDNAs were amplified by 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) Y is C or T, R is A or G, K is G or T, M is A or C.

The oligonucleotides NB151 and NB154 were used to amplify the 136 base pairs (bp) fragment to the 5 'end of the GHRH gene. NB152 and NB153 oligonucleotides were used to amplify an overlapping 206 bp fragment corresponding to the 3 'end of the GHRH gene.

100 µl reaction mixture contains 20 µ contains cDNA mixture, 2.5 units DNA polymerase (J. Cline et al. Nucl. Acid Res.1996, 24, 3546-3551 and H. Hogrefe et al. Proc. Natl. Acad. Sci USA 2002, 99, 596-601), 50 mM Tris HCl pH 8.2, 0.5 µg each oligonucleotide (NB151 / NB154 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 by the end of a 10 minute extension at 72 ° C.

The order of the nucleotide sequence of the 5 'and 3' ends of the GHRH mRNA was the amplified according to the RACE protocol. For the 5 'end a 5 µΙ mixture containing 3 µΙ of total hypothalamic RNA, 2 µΜ primary oligo dT 5'-CDS, 2 µΜ Smt oligonucleotide was heated at 70 ° C and chilled on ice for denaturation. 10 µl of reaction mixture containing 5 µl of denatured RNA with oligonucleotides, 50 mM Tris HCl pH 8.3, 75 mM KCl, 6 mM MgCl 2, 2 mM DTT, 1 mM each dNTP, 100 units reverse transcriptase 90 min at 42 ° C and then diluted 1:10 in a buffer 10mM Tricine-KOH pH 8.5, 1mM EDTA. 2.5 µΙ aliquot was used for amplification in a 50 µl reaction mixture containing also 0.04 µΜ Universal primer, 0.2 µΜ NB154 oligonucleotide, 40 mM Tricine-KOH pH 8.7, 15 mM KOAc, 3.5 mM Mg (OAc) 2, 3.75 µg / ml BSA , 0.005% Tween 20 ®, 0.005% Nonidet-P40, 0.2 µΜ each dNTP, 2.5 units Taq DNA polymerase. 5 cycl 3 minutes and 30 cycles of 94 ° C for 30 seconds, 65 ° C for 3 minutes, 72 ° C for 3 minutes. The 239 bp fragment was obtained.

Smt (30 mer) 5'-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3 '(SEQ ID NO: 9)

Oligo dT 5'-CDS 5 '- (T) 25 N 1 N-3', wherein N 1 is A, C or G and N is A, C, G or T.

Universal primer 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3 '(SEQ ID NO: 10) [0121] containing only 3 μΙ of total hypothalamic RNA, 2 μΜ SMART ( Clontech Laboratories, Palo Alto, California) oligonucleotide and the 50 µl reaction mixture for PCR containing 2.5 µΙ aliquot of diluted cDNA, 0.04 µΜ universal primer mix, 0.2 µΜ NB153 oligonucleotide, 50 mM Tris HCl pH 8.3, 75 mM KCl, 6 mM MgCl 2 , 3.75 µg / ml BSA, 0.005% Tween 20 ®, 0.005% Nonodet-P40, 0.2 µΜ each dNTP, 2.5 units Taq DNA polymerase. A 386 bp fragment was obtained. SMART (57 mer) 5'-AAGCAGTGGTATCAACGCAGAGTAC (T) 30N.1N-3 '(SEQ ID NO: 11), wherein N, is A, C or G and N is A, C, G or T.

The amplified fragments of the PCR were cloned directly into an appropriate plasmid. E. coli colonies were screened by colony lift hybridation. The plasmids with GHRH cDNA were identified by the plasmid pCRII NB151 / 154 and pCRII NB152 / 153. NB155 (50 mer) 5'-GCCATCTTCACYAACARCTACCGGAAGGTBCTGGGCCAGCTVTCYGCCCG-3 '(SEQ ID NO: 12), wherein 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 Following is the 5'-ATGCCACTCT GGGTGTTCTT CCTGGTGATC CTCACCCTCA GCAGTGGCTC CCACTCTTCC CCGCCATCCC TGCCCATCAG AATCCCTCGG TATGCAGACG CCATCTTCAC CAACAGCTAC CGGAAGGTGC TGGGCCAGCT GTCCGCCCGC AAGCTCCTGC AGGACATCAT GAGCCGGCAG CAGGGAGAGA GAAACCG-GGA GCAAGGAGCA AAGGTACGAC TCGGCCGTCA GGTGGACAGT CTGTGGGCAA GCCAAAAGCA GAT GGCATTG GAGAACATCC TGGCATCCCT GTTACAGAAA CGCAGGAACT CCCAAGGATG A-3 '(SEQ ID NO: 3). The corresponding amino acid sequence is the following: H-MPLWVFFLVILTLSSGSHSS PPSLPIRIPR YADAIFTN-SY RKVLGQLSAR KLLQDIMSRQ QGERNREQGA KVRLGRQVDS LWASQKQMAL ENILASLLQK RRNSQG-OH (SEQ ID NO: 1). The signal peptide spans from amino acid 1 to amino acid 20; the mature GHRH spans amino acid 31 to amino acid 74.

Example 2.

The polynucleotide encoding the canine GHRH is also obtained by RT-PCRfrom the total hypothalamic RNA using the NB 184 and NB 185 oligonucleotides and the 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 is its 5' end) NB185 (35 mer): 5'-AAAGCTCTAGATCAACGGCCGAGTCGTACCTTTGC-3 '(SEQ ID NO: 1) 14) contains Xba I site on its 5 'end.

The amplification is carried out with 1 cycle for reverse transcription step 42 ° C for 15 minutes, 95 ° C for 5 minutes, 4 ° C for 5 minutes and 30 cycles for PCR step 95 ° C for 45 seconds, 55 ° C for 45 seconds, 72 ° C for 1 minute.

The PCR product (164 pb) is also purified by phenol-chloroform extraction and digested by BamH I and Xba I to generate a BamH Ι / Xba I of 147 bp (fragment A).

The plasmid pAB110 is derived from pVR1020 plasmid (Vical Inc) by insertion of a polylinker (BamH I, Not I, Eco R I, Eco R V, Xba I, Pml I, Pst I, Bgl II) corresponding to the PB326 and PB329 oligonucleotides after BamH / Bgl II digestion of the plasmid. PB326 (40 mer) 5'-GATCTGCAGCACGTGTCTAGAGGATATCGAATTCGCGGCC-3 '(SEQ ID NO: 15) PB329 (40 mer) 5'-GATCCGCGGCCGCGAATTCGATATCCTCTAGACACGTGCT-3' (SEQ ID NO: 16) Plasmid pAB110 is linearized by a BamH Ι / Xba I digestion to generate fragment B (5055 bp).

Fragments A and B are ligated to generate plasmid pNB179 (5202 bp) (FIG. 1).

Example 3: The plasmid pCRII NB151 / 154 is first digested by EcoRI. The EcoRI fragment (167 bp) was digested by BsaWI to generate a EcoRI-BsaWI fragment (143 bp). The plasmid pCRII NB 152/153 is also fisrt digested by EcoRI. The EcoRI fragment (235 bp) is digested by BsaWI to generate a BsaWI-EcoRI fragment (220 bp).

These two fragments are plasmid pCRII NB151 / 154 linearized by EcoRI to generate the final plasmid pCRII NB151 / 152 (4295 bp).

Example 4: Construction of pNB209 is based on a pVR1012 plasmid (Vical Inc.) encoding the canine GHRH precursor (106 amino acid polypeptides).

The DNA fragment corresponding to the cGHRH gene, with additional 5 'and 3' ends, is amplified by PCR primers NB361 and NB360, the plasmid pCRIINB151 / 152 as a template and the DNA polymerase. NB360 (30 mer): 5'-AAAGCT CTAG AT CAT CCTT GGG AGTT CCT G-3 '(SEQ ID NO: 17) (contains a Xbal site on its 5' end) NB361 (31 mer): 5'-TTTACGCGTCGACATGCTGCTCTGGGTGTTC- 3 '(SEQ ID NO: 18) (contains a Sail site on its 5' end) The PCR fragment (345 bp) was purified by phenol-chloroform extraction and digested by Sail and XbaI to generate fragment (327). bp).

Plasmid pVR1012 is linearized by a SalI / XbaI digestion to generate fragment B (4880 bp).

Fragments A and B are ligated to generate plasmid pNB209 (5207 bp). FIG. 2 shows the plasmid map and the encoded ORF of pNB209.

Example 5: Construction of plasmids pNB210 / pNB211 / pNB212 / pNB213: pAB 110-derived plasmids containing modified GHRH genes, encoding hybrid proteins containing the eukaryotic tPA leader peptide fused to the GHRH propeptide.

The DNA fragment is as described by PCR using primers NB355 and NB356, plasmid pAB110 as a template and 5 'and 3' ends, respectively. the DNA Polymerase (J. Cline et al. 1996 and H. Hogrefe et al. 2002). NB355 (20 mer): 5'-CCGTCGTCGACAGAGCTGAG -3 '(SEQ ID NO: 19) (contains a Sail site of its 5' end) NB356 (24 mer): 5'-AAAAGCGCTGGGCGAAACGAAGAC-3 '(SEQ ID NO: 20) ) (contains a Eco47lll site is its 5 'end) The PCR fragment (184 bp) is also purified by phenol-chloroform extraction and digested by Sail and Eco471 II to generate fragment A (172 bp).

The DNA fragment is as described by PCR using primers NB355 and NB357, the plasmid pAB110 as a template. and the DNA Polymerase. NB355 (20 mer): 5'-CCGTCGT CGACAGAGCT GAG -3 '(SEQ ID NO: 19) (contains a Sail site on its 5' end) NB357 (42 mer): 5'-AAAGCCGGCATGGATTTCCTGGCTGGGCGAAACGAAGAC TGC-3 '(SEQ ID NO: 21) (contains the 5 AA additional of the tPA leader peptide and a Nael site is its 5 'end).

The PCR fragment (199 bp) was also purified by phenol-chloroform extraction and digested by Sail and Nael to generate fragment B (187 bp).

The DNA fragment with the deletion (1-19 AA) GHRH with additional NlalV and Xbal sites at, respectively, the 5 'and 3' ends, is obtained by PCR using primers NB358 and NB360, the plasmid pCRII NB151 / 152 as a template and the DNA Polymerase. NB358 (21 mer): 5'-TTTGGTTCCCCGCCATCCCTG-3 '(SEQ ID NO: 22) (contains a NlalV site on its 5' end) NB360 (30 mer): 5'-AAAGCT CT AG AT CAT CCTT GGG AGTT CCT G -3 '(SEQ ID NO: 17) (contains a Xbal site on its 5' end).

The PCR fragment (281 bp) was also purified by phenol-chloroform extraction and digested by NlalV and XbaI to generate fragment C (265 bp).

The DNA fragment corresponding to the deletion (1-20 AA) GHRH with additional Stul and Xball sites at, respectively, the 5 'and 3' ends, is obtained by PCR using primers NB359 and NB360, the plasmid pCRII NB151 / 152 as a template and the DNA Polymerase. NB359 (24 mer): 5'-TTTAGGCCTCCATCCCTGCCCATC-3 '(SEQ ID NO: 23) NB360 (30 mer): 5'-AAAGCTCTAGATCATCCTTGGGAGTTCCTG-3' (SEQ ID NO: 17) ) (contains a Xbal site on its 5 'end) The PCR fragment (278 bp) is also purified by phenol-chloroform extraction and digested by Stul and XbaI to generate fragment D (262 bp).

Plasmid pVR1012 is linearized by a SalI / XbaI digestion to generate fragment E (4880 bp).

[0146] Fragments E, A and C are ligated to generate plasmid pNB210 (5313bp). FIG. 3 shows the plasmid map and the encoded ORF of pNB210.

Fragment E, A and D are ligated to generate plasmid pNB211 (531 Obp). FIG. 4 shows the plasmid map and the encoded ORF of pNB211.

Fragment E, B and C are ligated to generate plasmid pNB212 (5331bp). FIG. 5 shows the plasmid map and the encoded ORF of pNB212.

4Fragment E, B, and are ligated to generate plasmid pNB213 (5328bp). FIG. 6 shows the plasmid map and the encoded ORF of pNB213. Example 6: Construction of Plasmids pNB214 / pNB215 Construction of plasmids pNB214 / pNB215: pAB110-derived plasmids containing modified GHRH genes, encoding hybrid proteins containing human PA fused to the canine GFIRFI mature peptide.

The DNA fragment corresponds to the peptide (31-74 AA) GFIRFI with additional Bst1107 and Xball sites at, respectively, the 5 'and 3' ends, also obtained by PCR using primers NB362 and NB363, the plasmid pCRII NB151 / The DNA Polymerase (J. Cline et al. 1996 and FI. Flogrefe et al. 2002). NB362 (27 mer): 5'-TTTGTATACGCAGACGCCATCTTCACC-3 '(SEQ ID NO: 24) (contains a Bst1107l site is its 5' end) NB363 (32 mer): 5'-AAAGCTCTAGATCAGAGTCGTACCTTTGCTCC-3 '(SEQ ID NO: 25) The PCR fragment (152 bp) is also purified by phenol-chloroform extraction and digested by Bst110711 and XbaI to generate fragment F (136 bp).

Fragment E, A and F are ligated to generate plasmid pNB214 (5184bp). FIG. 7 shows the plasmid map and the encoded ORF of pNB214.

Fragment E, B and F are ligated to generate plasmid pNB215 (5202bp). FIG. 8 shows the plasmid map and the encoded ORF of pNB215.

[0155] Fragment E, A and B are described in example 5.

Example 7: The canine pre-pro GFIRFI nucleotide sequence is optimized by removing cryptic splice sites, by adapting the codon usage to the one of Canis familiaris. SEQ ID NO: 40.

Construction of pNB216: is based on a pVR1012 plasmid (Vical Inc.) encoding the canine GFIRFI optimized precursor (106 amino acid polypeptides) of SEQ ID NO: 40.

The plasmid pCR-Script Amp-GFIRFI (Stratagene, Lajolla, CA, USA) inserting the optimized canine pre-proGFI-RFH gene digested with Sail and XbaI generate a SalI-XbaI fragment (336 bp) and plasmid pVR1012 is linearized by a SalI / XbaI digestion to generate fragment E (4880 bp). These two fragments are in the final plasmid pNB216 (5216 bp).

Example 8: Construction of plasmids pNB217 / pNB218: pAB110-derived plasmids containing modified GFIRFI genes encoding hybrid proteins containing the eukaryotic tPA leader peptide fused to the GFIRFI propeptide optimized.

The DNA fragment corresponding to the deletion (1-20 AA) GFIRFI optimized with additional NlalV and Xbal sites at, respectively, the 5 'and 3' ends, also obtained by PCR using primers NB364 and NB365, the pCR-Script Amp-GFIRFI plasmid as a template and the DNA Polymerase. NB364 (24 mer): 5'-TTTGGCCCCCCCAGCCTGCCCATC-3 '(SEQ ID NO: 45) (contains an NlalV site is its 5' end) NB365 (33 mer): 5'-AAAGCTCTAGATTATCAGCCCTGGCTGTTCCGC-3 '(SEQ ID NO: 46) ) (contains a Xbal site is its 5 'end) The PCR fragment (281 bp) is also purified by phenol-chloroform extraction and digested by NlalV and XbaI to generate fragment G (265 bp).

Plasmid pVR1012 is linearized by a SalI / XbaI digestion to generate fragment E (4880 bp).

Fragments E, A and G are ligated to generate plasmid pNB217 (5313bp). FIG. 10 shows the plasmid map and the encoded ORF of pNB217.

Fragment E, B and G are ligated to generate plasmid pNB218 (5328bp). FIG. 11 shows the plasmid map and the encoded ORF of pNB218.

Fragment A and B are described in example 5.

Example 9: Construction of plasmids pNB219 / pNB220: pAB110-derived plasmids containing modified GHRH genes, encoding hybrid proteins containing human tPA fused to the canine GHRH mature peptide optimized.

The DNA fragment corresponding to the peptide (31-74 AA) GHRH optimized with additional Bst1107I and Xbal sites at, respectively, the 5 'and 3' ends, is obtained by PCR using primers NB366 and NB367, the plasmid pCRII NB151 Polymerase (J. Cline et al. 1996 and H. Hogrefe et al. 2002). NB366 (27 mer): 5'-TTTGTATACGCCGACGCCATCTTCACC-3 '(SEQ ID NO: 47) (contains a Bst11071 site is its 5' end) NB367 (32 mer): 5'-AAAGCTCTAGATCACAGCCGCACTTTGGCGCC-3 '(SEQ ID NO: 48) ) (contains a Xbal site on its 5 'end) The PCR fragment (152 bp) is also purified by phenol-chloroform extraction and digested by Bst1107I and XbaI to generate fragment H (136 bp).

Fragment E, A and H are ligated to generate plasmid pNB219 (5184bp). FIG. 12 shows the plasmid map and the encoded ORF of pNB219.

Fragment E, B and H are plasmid pNB220 (5199bp). FIG. 13 shows the plasmid map and the encoded ORF of pNB220.

[0171] Fragment A and B are described in example 5.

Example 10: Method for low voltage electroporation for DNA uptake and expression in an animal. Patent Publication 20040057941.

[0173] The method described herein can be modified for the present description without undue experimentation.

Direct intra-muscular plasmid DNA injection by electroporation is a method of local and controlled delivery of plasmid DNA into skeletal muscle. It has the advantage that it uses low plasmid quantities (as low as 0.1 mg). Although not wanting to be bound by theory, it is also possible to carry out the process of transporting cells with or without protein active transport. Although not wanting to be bound by the theory of the field of energy (Bureau et al., Biochim Biophys Acta. 2000 May 1; 3): 353-9 and Gilbert et al., Biochim Biophys Acta 1997 Feb 11; 1334 (1): 9-14.) And cell size (Somiari et al., Mol Ther. 2000 Sep; 2 (3)) : 178-87). Classical electrode configuration, plate ora pair of wire electrodes placed in 4 mm apart. These are the types of electrodes unpractical. The porcine or dog muscle fibers are quite large and suitable for electropenneabilization than rodent muscle. This article was previously published under Q399260 Copyright © 2006 Nokia. All rights reserved. , allow the subject to gain weight, and extend life expectancy of the chronically;

The pSP-H V-G H RH system (the vector expressing porcine GH RH) was provided to the left tibial anterior m uscle of healthy dogs via in vivo electroporation. A group of 4 dogs (2 males and 2 females) were injected with the pSP-HV-GHRH system. The dogs were injected with vehicle alone, or 200mcg, or 600mcg or 100Omcg of pSP-HV-GHRH followed by needle electroporation. An indication of GHRH and GH is an increase in serum IGF-I concentration. Therefore, the following days, the injection was injected with vehicle alone, or 200mcg or 1000mcg of pSP-HV-GHRH and IGF-I levels were determined. The IGF-I levels were injected with 600 µl of vehicle alone treated animals (FIG. 2). The increase in IGF-I levels was statistically significant (p <0.046). And injected with 200 µg and 1000 µg of plasmid showed higher IGF-I levels were injected with 600 µl. Increased IGF-I levels in GHR (utilized recombinant porcine GH ("pGH") in dogs. For example, there were dose-related increases in serum IGF-I levels (approximately 2-10 fold) in pGH treated dogs.

But not wanting to be bound by theory, growth hormone releasing hormone (GHRH) stimulating the production and release of the anterior pituitary of growth hormone (GH), which in turn stimulates the production of IGF-I from the liver and other target organs. Thus, an increase in serum IGF-I concentration. The levels of serum IGF-I in healthy dogs injected with 200, 600 and 1000 µg of pSP-HV-GHRH were all 28 days post-injection when compared to the pre-injection values. Dogs injected with 600 and pSP-HV-GHRH showed the highest statistically significant increase (e.g., greater than 90%, p <0.046) in IGF-I levels, which indicates that 600% may be the optimal concentration for healthy dogs.

Example 11: Construction of plasmids pNB240, pNB239 and pNB244.

Construction of pNB240 is based on plasmid pVR1012 (Vical Inc.) as modified GHRH gene encoding the hybrid protein containing the pre-proGHRH deleted C-terminus and fused to the c-myc epitope tag.

Constructs of pNB239 and pNB244 are based on the plasmid pVR1012 (Vical Inc.), modified GHRH gene encoding the hybrid protein containing the equine IGF1 (Insulin-like Growth Factor 1) peptide signal sequence (SSIGF1, disclosed in Genbank). under the 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 C-terminus of the propeptide (corresponding to 1-74 AA) and fused to the c-myc epitope tag, with additional Xbal site at the 3 'end, is amplified by PCR using primers PB091 and NB412, the plasmid pNB209 (cf. example 4) as a template and the DNA Polymerase. PB091 (22 mer): 5'- CCAGACATAATAGCTGACAGAC -3 '(SEQ ID NO: 51)

NB412 (68 mer): 5'-AAAG CT CTAG ATTAG AG AT CCT CTT CT GAG AT AAGCTTTT GTT CG AGT CGT ACCTT TGCTCCTTGCTC -3 '(contains the c-myc epitope tag and a Xbal site on its 3' end) (SEQ ID NO: 52) The PCR fragment (338 bp) was purified by phenol-chloroform extraction and digested by Sail and XbaI to generate fragment A (261 bp).

The equine pre-pro IGF1 nucleotide sequence is optimized by removing cryptic splice sites, by introducing a Kozak consensus sequence before the start codon in order to improve expression. SEQ ID NO: 65.

The plasmid pCR-Script Amp-elGF1 (Stratagene, Lajolla, CA, USA) contains the optimized equine IGF1 gene used as a template for PCR primers NB393 and NB394 and the DNA Polymerase. NB393 (38 mer): 5'- TTTGCGTCGACGCCACCATGCACATCATGAGCAGCAGC -3 '(contains a Sail site on its 5' end) (SEQ ID NO: 66) NB394 (38 mer): 5'- AAAGCTCTAGATTATCACATCCGGTAGTTCTTGTTGCC -3 '(contains a Xbal site is its 5 'end (SEQ ID NO: 67) The PCR fragment (424 bp) is also purified by phenol-chloroform extraction and digested by Sail and XbaI to generate fragment F (408 bp).

Plasmid pVR1012 is linearized by a SalI / XbaI digestion to generate fragment E (4880 bp).

[0185] Fragments E and F are ligated to generate plasmid pNB231 (5288bp).

The DNA fragment corresponds to the equine SSIFG1 (1-25 AA of FIGS. 15-18) with additional Nael site at the 3 'end, obtained by PCR using primers PB091 and NB398, plasmid pNB231 as a template and the DNA Polymerase (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 is its 3' end) (SEQ ID NO: 53) The PCR fragment ( 159 bp) is also purified by phenol-chloroform extraction and digested by Sail and Nael to generate fragment B (86 bp).

The DNA fragment corresponding to the GHRH mature peptide (31-74 AA) fused to the c-myc epitope tag, with additional 5 'and 3' ends, respectively, by PCR using primers NB362 and NB412, 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 also purified by phenol-chloroform extraction and digested by Bst1107l and Xbal to generate fragment C (166 bp).

The DNA fragment is the C-terminus of the GHRH pro-peptide deleted from the propeptide (20-74 AA) fused to the c-myc epitope tag, with additional 5 'and 3, respectively. 'ends, also obtained by PCR using primers NB358 and NB412, 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 also purified by phenol-chloroform extraction and digested by Bst1107I and Xbal to generate fragment D (199 bp).

Plasmid pVR1012 is linearized by a SalI / XbaI digestion to generate fragment E (4880 bp).

[0193] Fragments E and A are and are ligated to generate plasmid pNB240 (5141bp). FIG. 14 shows the plasmid map and the encoded ORF of pNB240.

Fragment E, B and C are ligated to generate plasmid pNB239 (5132bp). FIG. 15 shows the plasmid map and the encoded ORF of pNB239.

Fragment E, B, and are ligated to generate plasmid pNB244 (5165bp). FIG. 16 shows the plasmid map and the encoded ORF of pNB244.

The in vitro expressions of the fusion proteins encoded by pNB239, pNB240, and pNB244 were studied after transient transfection of CHO-K1 cells (available prior to the American Tissue Culture Collection, No. CCI-61). ), using Lipofectamine 2000. CHO-K1 cells at 90% confluency in 6 cm diameter plates were transfected with 5 µg of plasmid and 10 μΙ lipofectamine each, according to manufacturer's instructions. After transfection, cells were cultivated in MEM-glutamate medium containing 1% FCV (fetal calf serum) for 24 hours. Culture supernatants were harvested and concentrated 40 times by trichoroacetic acid precipitation of proteins. Cells were washed with PBS, harvested by scraping, and lyzed using Laemmli SDS-PAGE loading buffer. Recombinant protein production and secretion (40 x) culture supernatants to SDS-PAGE and Western blotting using an anti-c-myc monoclonal antibody (Euromedex, France).

Plasmid pNB240 encodes a hybrid protein containing aminoacids 1 to 74 of the preproGHRH and fused to the c-myc epitope tag.

Plasmid pNB239 encodes the hybrid protein containing the equine IGF1 peptide signal sequence (SSIGF1) fused to the GHRH mature peptide (31-74 AA) and the c-myc epitope tag.

Plasmid pNB244 encodes the hybrid protein containing the equine IGF1 peptide signal sequence (SSIGF1) fused to the GHRH propeptide (20-74 AA) and the c-myc epitope tag.

Expression results are summarized in the Table 1. The three constructs of the recombinant hormones.

Table 1:

Example 12: Construction of plasmids pNB232 and pNB245 Constructions of pNB232 and pNB245 are based on plasmid pVR1012 (Vical Inc.), modified GHRH gene encoding the hybrid protein containing the equine IGF1 peptide sequence signal (SSIGF1, detected in). Genbank under the accession numbers U28070 (24-48 AA) and U85272) fused to the modified GHRH.

The DNA fragment is the N-terminus of the propeptide GHRH deleted of the propeptide (corresponding to 31-106 AA) with additional Bst1107 and XbaI sites, respectively, by PCR using primers NB362 and NB421, plasmid pNB209 (see Example 4) as a template and the DNA Polymerase (J. Cline et al. 1996 and H. Hogrefe et al. 2002). NB362 (27 mer) (SEQ ID NO: 24) NB421 (36 mer): 5'-AAAGCTCTAGATTATCCTTGGGAGTTCCTGCGTTTC-3 '(contains a Xbal site on its 5' end) (SEQ ID NO: 54) The PCR fragment ( 248 bp) is also purified by phenol-chloroform extraction and digested by Bst1107I and XbaI to generate fragment G (232 bp).

Plasmid pVR1012 is linearized by a SalI / XbaI digestion to generate fragment H (4880 bp).

Fragment], B and F are ligated to generate plasmid pNB232 (5102bp). FIG. 17 shows the plasmid map and the encoded ORF of pNB232.

Fragment], B and G are ligated to generate plasmid pNB245 (5198bp). FIG. 18 shows the plasmid map and the encoded ORF of pNB245.

11. The fragment F is described in example 6.

Example 13: Construction of plasmids pNB228, pNB297 and pNB298 The plasmid pNB228 is the plasmid pVR1012 (Vical Inc.) backbone containing the nucleic acid fragment encoding the preproGHRH peptide deleted of the carboxy-terminal propeptide.

Plasmid pNB297 to the pVR1012 plasmid (Vical Inc.) backbone containing the nucleic acid fragment encoding the amino acids 1 to 74 of the preproGHRH and glycine at its carboxy-terminal end. The carboxy-terminal glycine should be easily amidated by amidation enzyme.

Plasmid pNB298 to the pVR1012 plasmid (Vical Inc.) backbone containing the nucleic acid fragment encoding the equine IGF1 signal sequence (SSIGF1) fused to the mature GHRH (31-74 AA) and a glycine at its carboxy terminal end.

The DNA fragment is to the preproGH RH deleted from the carboxy-terminal propeptide, with additional PCR using the primers NB361 and NB363, the plasmid pNB209 as a template and the DNA polymerase. NB361 (31 mer): 5'- TTTACGCGTCGACATGCTGCTCTGGGTGTTC -3 '(SEQ ID NO: 17) (contains a Sail site on its 5' end) NB363 (32 mer): 5'-AAAGCTCTAGATCAGAGTCGTACCTTTGCTCC-3 '(SEQ ID NO: 24) ) (contains a Xbal site on its 5 'end) The PCR fragment (249 bp) is also purified by phenol-chloroform extraction and digestion by Sail and XbaI to generate the fragment A (231 bp).

The DNA fragment of the preproGHRH (1-74 AA) and glycine at its carboxy-terminal end, with additional 5 'and 3' ends, respectively, is amplified by PCR using the primers NB361 and NB476, plasmid pNB209 as a template and the DNA polymerase. NB361 (31 mer): 5'- TTTACGCGTCGACATGCTGCTCTGGGTGTTC -3 '(SEQ ID NO: 17) NB476 (32 mer): 5'-AAAGCTCTAGATTAGCCGAGTCGTACCTTTGC -3' (SEQ ID NO: 68) The PCR fragment (252 bp) is also purified by phenol-chloroform extraction and digested by Sail and XbaI to generate the fragment B (234 bp).

The DNA fragment is the equivalent of the IGF1 signal sequence (SSIGF1) fused to the mature GHRH (31-74 AA) and glycine at its carboxy-terminal end, with additional 5 'and 3' ends , respectively, is also 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 (38mer): 5'- TTTGCGTCGACGCCACCATGCACATCATGAGCAGCAGC -3 '(SEQ ID NO: 66) NB476 (32 mer): 5'- AAAGCT CTAG ATTAG CCG AGT CGT ACCTTT GC -3' (SEQ ID NO: 68) (contains a Xbal site on its 5 'end) The PCR fragment (241 bp) is also purified by phenol-chloroform extraction and digestion by Sail and XbaI to generate the fragment C (225 bp) ).

The plasmid pVR1012 is linearized by a SalI / XbaI digestion to generate the fragment E (4880 bp).

Fragments E and A are and are ligated to generate the plasmid pNB228 (5111 bp). FIG. 19 shows the plasmid map and the encoded ORF of pNB228.

Fragment E and B are ligated to generate the plasmid pNB297 (5114bp). FIG. 20 shows the plasmid map and the encoded ORF of pNB297.

Fragment E and C are ligated to generate the plasmid pNB298 (5105bp). FIG. 21 shows the plasmid map and the encoded ORF of pNB298.

Plasmids similar to plasmids pNB297 and pNB298 containing the porcine GHRH (SEQ ID NO: 82) instead of canine GHRH sequence according to the method described in Example 13 and examples, are: NB464 (37 mer). 27 mer) 5'- TTTGTATACGCCGACGCCATCTTCACC -3 '(SEQ ID NO: 85) corresponding to the primer NB362 and NB418 (38 mer) 5'-AAAGCTCTAGATTACAGCCTCACCCTGGCGCCCTGCTC-3' (SEQ ID NO: 86) corresponding to the primer NB363 NB465 (39 mer) (SEQ ID NO: 84) corresponding to the primer NB476.

The porcine GHRH (231 mer) (SEQ ID NO: 82): 5'-ATGCTGCTGTGGGTGTTCTTCCTGGTGACCCTGACCCT GAGCAGCGGCAG CCTGAGCAGCCTGCCCAGCCAGCCCCTGAGGATGCCCAGGTACGCCGACG COAT-C ACC CTT AAC AG CT AC G AG AAG GTG GAG CTG GG CC CG CT AG CCAG AAGCTGCTGCAGGACATCAT-G GA CAG AAC GAGCAGGCAGCAGGGCGAGAGG GCAGGGCGCCAGGGTGAGGCTGGGCAGGCAGGT -

GGACAGCATGTGGGCCG ACCAG AAG CAG ATG GCCCTGGAGAGCATCCTGGCCACCCT GCTGCAGGAG C AC AG G AAC AG CCAG G G CTG A -3 '.

Example 14: Construction of plasmids pNB299 and pNB300 The pNB299 plasmid is the plasmid pVR1012 (Vical Inc.) backbone containing the nucleic acid fragment encoding the equine IGF1 signal sequence (SSIGF1) fused to the mature GHRH (31-74 AA) ).

The pNB300 plasmid to the pVR1012 plasmid (Vical Inc.) backbone containing the nucleic acid fragment encoding the equine IGF1 signal sequence (SSIGF1) fused to the mature GHRH (amino acids 31-74 of the precursor) and the canine serum albumin (amino acids 25 to 608 of the precursor) through a linker tetrapeptide (GSGS).

The DNA fragment with the equine IGF1 signal sequence (SSIGF1) fused to the mature GHRH (31-74 AA), with additional Sail 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 (38mer): 5'- TTTGCGTCGACGCCACCATGCACATCATGAGCAGCAGC -3 '(SEQ ID NO: 17) NB477 (50 mer): 5'-AAATCTAGAGCCGGTTTAAAAGACCGTAGTCGTACCTTTGCTCCTTGCTC-3' (SEQ ID NO: 75) (contains a XbaI and PshAI sites are its 5 'end) The PCR fragment (250 bp) is also purified by phenol-chloroform extraction and digestion by Sail and XbaI to generate the fragment (236 bp).

The plasmid pVR1012 is linearized by a SalI / XbaI digestion to generate the fragment E (4880 bp).

Fragments E and A are and are ligated to generate the plasmid pNB299 (5116bp). FIG. 22 shows the plasmid map and the encoded ORF of pNB299.

The DNA fragment was linked to the linker GSGS fused to the canine serum albumin (amino acids 25 to 608), with additional 5 'and 3' ends, respectively, obtained by RT-PCR.

[0229] Liver is collected from a liquid nitrogen. Total RNAs are extracted from the tissue from a QIAGEN (Rneasy Mini Protocol for Isolation of Total RNA Catalog ref. 74104). The cells are dissociated with a pottery of 600 µΙ of denaturing solution from the kit. This solution contains guanidinium isothiocyanate and beta-mercaptoethanol. The tissue homogenate is centrifuged for 5 minutes at 14,000 RPM (rotations per minute) to remove debris. 600μΙ of a 70% ethanol solution is added and the mixture is loaded onto a Rneasy colomn and centrifuged for 15 seconds at 10,000 RPM. The RW1 buffer provided with the kit.

The RNA was also eluted with 50 μΙ RNAse-free buffer after centrifugation 1 minute at 14,000 RPM. The cDNAs are synthesized in a 20 µl reaction mixture containing 5 mM MgCl 2, 20 mM Tris HCl pH 8.3, 100 mM KCl, 1 mM DTT, 1 mM each dNTP, 20 units RNAse inhibitor, 50 units Moloney murine leukemia virus reverse transcriptase, 2.5 μΜ random hexanucleotide primers and 2 μΙ of total RNA from the liver. The reverse transcription step is done 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 cDNAs are amplified by Polymerase Chain Reaction (PCR) using the DNA polymerase and the following oligonucleotides: NB478 (43 mer): 5'-TTTGCCGGCTCAGGATCCGAAGCATATAAGAGTGAGATTGGTC-3 '(SEQ ID NO: 78) and the linker GSGS is its 5 'end) NB479 (35 mer): 5'-AAAGCTCTAGATTAGACTAAGGCAGCTTGAGCAGC-3' (SEQ ID NO: 79) (contains a Xbal site on its 5 'end) The PCR fragment (1784 bp) ) is also analyzed by phenol-chloroform extraction and digested by Nael and Xbal to generate the fragment B (1768 bp).

The plasmid pNB299 is linearized by a PshAI / XbaI digestion to generate the fragment C (5098 bp).

Fragments C and B are the ligated to generate the plasmid pNB300 (6866bp). FIG. 23 shows the plasmid map and the encoded ORF of pNB300.

Plasmids similar to plasmids pNB299 and pNB300 containing the porcine GHRH (SEQ ID NO: 82) instead of canine GHRH sequencing, are constructed by: NB459 (53 mer) 5 '-AAATCTAGAGCCGGTTTAAAAGACCGTAGTCTCACCCTGGCGCCCTGCTCCTG-3' (SEQ ID NO: 87) corresponding to the primer NB477. NB457 (40 mer) 5'-TTTGCCGGCTCAGGATCCGATACATACAAGAGTGAAATTG-3 (SEQ ID NO: 88) corresponds to the primer NB478 and NB458 (29 mer) 5'-AAAGCTCTAGATTAGGCTAAGATCCCTCG -3 '(SEQ ID NO: 89) corresponding to the primer NB479.

Claims 1. An expression vector is a polynucleotide that encodes a hybrid protein containing equine IGF-1 signal peptide fused to canine mature GHRH and a c-myc epitope tag, which is a promoter, and promoter, and and the amino acid sequence shown as amino acids 31-74 of SEQ ID NO: 1 (page 7, paragraph [0049], and pages 31-32, paragraph [0136]). 2. The hybrid protein is the amino acid sequence shown as SEQ ID NO: 58 (page 6, paragraph [0039], and Figure 15, page 15/23). 3. An expression vector polynucleotide that encodes a hybrid protein containing equine IGF-1 signal peptide fused to canine proGHRH and a c-myc epitope tag, which is a promoter, and a promoter, and canine proGHRH has the amino acid sequence shown as amino acids 20-74 of SEQ ID NO: 1 (page 7, paragraph [0049], and pages 31-32, paragraph [0136]). 4. The expression vector amino acid sequence was shown as SEQ ID NO 60 (page 6, paragraph [0040], and Figure 16, page 16/23). 5. The formulation for delivery and expression of a canine mature GHRH in a cell, which is a vector or claim or vehicle or excipient. 6. A formulation for delivery and expression of a canine proGHRH in a cell, which is a vector or claim or vehicle or excipient. 7. The formulation of claim 5, which is the carrier or excipient facilitation transfection and / or improvisation of the vector or protein. 8. The formulation of claim 6, or the vehicle or excipient facilitation transfection and / or improvisation of the vector or protein. Use of an expression vector for any one of the claims 1 to 4, or a method of treating anemia, cachexia, obesity or osteoporosis in a vertebrate. 10. An expression vector of one of claims 1 to 4, or a method of treatment of anemia, cachexia, obesity or osteoporosis in a vertebrate. 11. The vertebrate is a cattle, dog or pig. 12. The vertebrate is a cattle, dog or pig.

Patent No. 1: Expression vector, der ein Polynucleotide umfasst, das ein Hybrid protein codiert, das ein IGF-1-Signalpeptide vom Pferd enthalt, defin gif HGHH vom Hund und c-myc-Epitop-Tag fusioniert ist, wobei das Polynucleotid mit einem Enhancer und / oder Promoter function of the transgene and Wo rd d a re re GHRH vom Hund die als Aminosaur 31-74 von SEQ ID NO: 1 Genesis Amenzauresequenz hat (Seite 7, Absatz [0049] and Seiten 31-32, Absatz [0136]). 2. Expression vector nach Anspruch 1, wobei das Hybrid protein die als from SEQ ID NO: 58 from Aminosauresequenz umfas (Seite 6, Absatz [0039] and Fig. 15, Seite 15/23). 3. Expression vector, der ein Polynucleotide umfasst, das ein Hybrid protein codiert, iGF-1-Signalpeptide vom Pferd enthalt, which is a proGHRH vom Hund and not c-myc-Epitop-Tag fusioner, wobei das Polynucleotid mit einem Enhancer und / oder Promoter functional network and proGHRH vom Hund die als Aminosaur 20-74 von SEQ ID NO: 1 genesis Aminosauresequenz hat (Seite 7, Absatz [0049] and Seiten 31-32, Absatz [0136]). 4. Expression vector nach Anspruch 3, wobei das Hybrid protein die als from SEQ ID NO: 60 from Aminosauresequenz umfas (Seite 6, Absatz [0040] and Fig. 16, Seite 16/23). 5. Formulierung fur die Abgabe (Delivery) and Expression Erythrocyte GHRH vom Hund in einer Zelle, wobei die Formulierung den Vector nach Anspruch 1 oder2 and et al pharmazeutisch Oder veterinarmedizinisch vertraglichen Tragerstoff or Exzipienten umfasst. 6. Formulerung fur die Abgabe and Expression Probe for proGHRH vom Hund in einer Zelle, wobei die Formulierung Vector nach Anspruch 3 Oder 4 and ein pharmazeutisch Oder veterinarmedizinisch vertraglichen Trager, Tragerstoff Oder Exzipienten umfasst. 7. Formulierung nach Anspruch 5, Warte der Trager, TragerstofFoder Exzipient die transfection, and / or die Erhaltung des vector Oder Protein verbesser. 8. Formulierung nach Anspruch 6, Warte der Trager, Tragerstoff Oder Protein Transfection, and / or Die Erhaltung des Vectors Oder Protein Verbesser. 9. Verwendung eines Expression vector nach einem der Anspmche 1 bis 4 Oder einem Formulierung nach Anspruch 5 Oder 6 zur Herstellung eines Medik fur die Behandlung von Anamie, Kachexie, Adiposit Oder Osteoporose in einem Wirbeltier. 10. Expressionsvector nach einem derAnspruche 1 bis 4 Oder Formulierung nach Anspruch 5 Oder 6 fur die verwendung der der Behandlung von Anamie, Kachexie, Adiposit Oder Osteoporose in einem Wirbeltier. 11. Verwendung nach Anspruch 9, wobei das Wirbeltier ein Rind, Hund or Schwein. 12. Expression vector or Formulierung zur Verwendung nach Anspruch 10, wobei das Wirbeltier ein Rind, Hund Oder Schwein.

Revendications 1. Vecteur d'expression comprenant and polynucléotide qui code pour une protéine hybride contenant le peptide de l'IGF-1 ququin fusionné la la la Gordon cine myc, dans lequel le polynucléotide est lié de façon fonctionnelle à and amplificateur et / ou and promotur et dans lequel la Gene H and canine a la séquence d'acides amine and para-aces amine 31 à 74 de SEQ ID NO: 1 (page 7, para. par [0049] to pages 31-32 , para-paragraph [0136]). 2. Vecteur d'expression selon la revendication 1 dans lequel laden protéine hybrids comprend la séquence d'acides amine représentée par SEQ ID NO: 58 (page 6, para., Para. 15, page 15/23). 3. Vecteur d'expression comprenant and polynucléotide qui code pour une protéine hybride contenant le peptide de l'IGF-1 etquin fusionn la la proGHRH canine et un marqueur ehitopique c-myc, dans lequel le polynucléotide est lié de façon fonctionnelle à and amplifiers et / ou and promoter and dans lequel la proGHRH canine a la séquence d'acides amine représentée par les acides amine 20 à 74 de SEQ ID NO: 1 (page 7, para. par. 0136]). [0040] 4. Vecteur d'expression selon la revendication 3 dans lequel laden protéine hybrids comprend la séquence d'acides amine représentée par SEQ ID NO: 60 (page 6, para. Par., P. 16, page 16/23). 5. Formulation pour l'administration et l'expression d'une GHRH mature canine dans une cellule, dans laquelle la formulation and support, and vice versa pharmaceutique ou vétérinaire. 6. Formulation pour l'administration et l'expression d'une proGHRH canine dans une cellule, dans laquelle la formulation comprend le vecteur selon la revendication 3 ou et et al. ou vétérinaire. 7. Formulation selen la revendication 5, dans laquelle le support, le véhicule ou l'excipient facilite la transfection et de ou amélore la conservation du vecteur ou de protéine. 8. Formulation selen la revendication 6, dans laquelle le support, le véhicule ou l'excipient facilite la transfection et de ou amélore la conservation du vecteur ou de protéine. 9 de la cachexie 9. Or. De la cachexie, de la cachexie. , de l'obésité ou de l'ostéoporose chez un vertébré. 10 vecteur d'expression selon l'une quelconque des revendications 1 o 4, ou formulation selon la revendication 5 ou 6 pour une utilisation dans le traitement de l'anemie, de la cachexie, de l'obésité ou de l'ostéoporose chez un vertébré. 11. Utilization of revitalization 9, dans laquelle le vertébré est and bovin, and chien ou and porc. 12. Vecteur d’expression ou formulation pour une utilisation selon la revelation le din le le dé leé and bovin, and chien ou and porc.

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Claims (5)

1. Fxpsvikski vekiog, IÉÍML sgiplipgptkfet keii slsfei OHR | l ~ | ÉÉóz,:, and c-mya ephop labeled by hybrid, where the polyvklvolid sbÜkIkIe is capable of e??? vaa, and where &amp; katpi? á ^! É «..! SMMIéaía.jaftss.v-s« #! p8ciájá ·, · »::% m. I »»: i all over! 31 -24. aabaosavakí ?. okiak pbük) swing M ~ m. ma% A® i ... sgenypoat serbit expressi vector where itibdd's lambs are lambs in the SIQ 10 MO 51 saeebali skiing (s): (b> ok, p039j and 1 s, 13/23) page). 3, ExprossAa vector containing α-α-β-κK-βK-α, α-βp-β-κK-β, and s-βp-β-α-α-α α-α va va va va va va va va va va va va va va va va. containing hybrids:,, where the polyglycotomy moxbase suffers to an enoserus and / or protease, and wherein the pro-α1-llk3 of agaric acid is agarose: sSoO 10140:} serbite: 20-74. ammo acids (?, ol4s | [8Ö49. | triggers a3i, -32 :, pl3a,}; 4, A 3:. Claims Sgtae Expression Vector, where the hybrid retrieval is provided »Amino acid scavenging according to SEQ 1D NO. | b «kea £ ÍáS: and lo> Figure,. 16/23. side),. 5, a preparation for ripe dog Ok for Mklf cell proliferation and cellular expansion, wherein the composition comprises claims 1 or 2; zgidöd :: and: gurges: liag or animal gonadsddbaBo 'hoTdoza, a substance or auxiliary substance of the invention. % &amp; Incense cybercrime &amp; cellular cellular dermatology and cellular 11 expresazky, where the knife or astringent orchestrate and germinating or grafting or doping of the cellar, or yoga, or timeP &amp; eyapo 7, Áx 5 .. jományomom series, the carrier, carrier or excipient promotes lrastsa; 8, A. A kit according to claim 1, wherein the carrier, vivdaayag or excipient promotes the shelf life of the actuator or enhancer. A The M.> Ye-Bean, Bovine, Serial Expressive Vector, or ®: A. or 4, ·. The use of a composition according to the invention is intended for the manufacture of a medicament for the treatment of anemia, oachexia, obesity or osteoporosis. horse. The expression vector of any one of Claims 1-4, Eyes, or 8¾ A or A, is a composition for treating vertebrate anemia, easel, sciatica, or osteeporex: for use in tetenf. 1 1, A, A claim set in the Gelsexar Saryanar, or a dAanar 12. A serotype expression vector of claim 10, wherein the vertebrate cattle, lice or pig are used.