WO2002061037A2 - Administration d'une sequence d'acides amines a un animal femelle - Google Patents

Administration d'une sequence d'acides amines a un animal femelle Download PDF

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Publication number
WO2002061037A2
WO2002061037A2 PCT/US2001/048726 US0148726W WO02061037A2 WO 2002061037 A2 WO2002061037 A2 WO 2002061037A2 US 0148726 W US0148726 W US 0148726W WO 02061037 A2 WO02061037 A2 WO 02061037A2
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Prior art keywords
animal
vector
female
cells
offspring
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PCT/US2001/048726
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English (en)
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WO2002061037B1 (fr
WO2002061037A3 (fr
Inventor
Robert J. Schwartz
Robert H. Carpenter
Ruxandra Draghia-Akli
Douglas R. Kern
Roy G. Smith
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Baylor College Of Medicine
Advisys, Inc.
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Application filed by Baylor College Of Medicine, Advisys, Inc. filed Critical Baylor College Of Medicine
Priority to KR10-2003-7007872A priority Critical patent/KR20040039187A/ko
Priority to CA2430921A priority patent/CA2430921C/fr
Priority to MXPA03005236A priority patent/MXPA03005236A/es
Priority to EP01997073A priority patent/EP1364004A4/fr
Priority to AU2002248194A priority patent/AU2002248194B2/en
Priority to BR0116472-4A priority patent/BR0116472A/pt
Publication of WO2002061037A2 publication Critical patent/WO2002061037A2/fr
Publication of WO2002061037A3 publication Critical patent/WO2002061037A3/fr
Publication of WO2002061037B1 publication Critical patent/WO2002061037B1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0271—Chimeric animals, e.g. comprising exogenous cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/60—Growth-hormone releasing factors (GH-RF) (Somatoliberin)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • This invention relates generally to endocrinology, medicine and cell biology. More specifically, the invention relates to the improvement of growth and performance; the stimulation of production of growth hormone in an animal at a level greater than that associated with normal growth; and the enhancement of growth utilizing the administration of DNA encoding a growth hormone releasing hormone into a female animal. Furthermore, it relates to the application of a nucleotide sequence that enhances growth, such as growth hormone releasing hormone or an analog, regulated by a muscle-specific promoter into muscle tissue, particularly using electroporation techniques.
  • the growth hormone (GH) production pathway is composed of a series of interdependent genes whose products are required for normal growth.
  • the GH pathway genes include: (1) ligands, such as GH and insulin-like growth factor-I (IGF-I); (2) transcription factors such as prophet of pit 1, or prop 1, and pit 1; (3) agonists and antagonists, such as growth hormone releasing hormone (GHRH) and somatostatin, respectively; and (4) receptors, such as GHRH receptor (GHRH-R) and the GH receptor (GH-R).
  • IGF-I insulin-like growth factor-I
  • GHRH growth hormone releasing hormone
  • GHRH-R GHRH receptor
  • GH-R GH receptor
  • GH pathway Effective and regulated expression of the GH pathway is essential for optimal linear growth, as well as homeostasis of carbohydrate, protein, and fat metabolism GH synthesis and secretion from the anterior pituitary is stimulated by GHRH and inhibited by somatostatin, both hypothalamic hormones.
  • GHRH GHRH
  • somatostatin both hypothalamic hormones.
  • the central role of GH in controlling somatic growth in humans and other vertebrates, and the physiologically relevant pathways regulating GH secretion from the pituitary are well known.
  • GH increases production of IGF-I, primarily in the liver, and other target organs. IGF-I and GH, in turn, feedback on the hypothalamus and pituitary to inhibit GHRH and GH release.
  • GH has both direct and indirect actions on peripheral tissues, the indirect effects being mediated mainly by IGF-I.
  • IGF-I insulin growth factor-I
  • Non-GH-deficiencies have different etiology, such as: (1) genetic diseases, Turner syndrome (Jacobs et al., 1990; Skuse et al., 1999), hypochondroplasia (Tanaka et al, 1998; Key and Gross, 1996), and Crohn's disease (Savage et al., 1999); and (2) intrauterine growth retardation (Albanese and Stanhope, 1997; Azcona et al, 1998); and (3) chronic renal insufficiency (Sohmiya et al, 1998; Benfield and Kohaut, 1997).
  • GH replacement therapy is widely used in patients with growth deficiencies and provides satisfactory growth, and may have positive psychological effects on the children being treated (Rosenbaum and Saigal, 1996; Erling, 1999), this therapy has several disadvantages, including an impractical requirement for frequent administration of GH (Monti et al., 1997; Heptulla et al., 1997) and undesirable secondary effects (Blethen et al, 1996; Watkins, 1996; Shalet et al, 1997; Allen et al, 1997).
  • GHRH extracranially secreted GHRH
  • mature peptide or truncated molecules as seen with pancreatic islet cell tumors and variously located carcinoids
  • carcinoids are often biologically active and can even produce acromegaly
  • Administration of recombinant GHRH to GH-deficient children or adult humans augments IGF-I levels, increases GH secretion proportionally to the GHRH dose, yet still invokes a response to bolus doses of GHRH (Bercu and Walker, 1997).
  • GHRH administration represents a more physiological alternative of increasing subnormal GH and IGF-I levels (Corpas et al, 1993).
  • GHRH protein therapy entrains and stimulates normal cyclical GH secretion with virtually no side effects
  • the short half-life of GHRH in vivo requires frequent (one to three times a day) intravenous, subcutaneous or intranasal (requiring 300- fold higher dose) administration.
  • GHRH administration is not practical.
  • extracranially secreted GHRH as a processed protein species (Tyr 1-40 or Tyrl-Leu44) or even as shorter truncated molecules, are biologically active (Thomer et al, 1984).
  • GHRH a low level of GHRH (100 pg/ml) in the blood supply stimulates GH secretion (Corpas et al, 1993) and makes GHRH an excellent candidate for gene therapeutic expression.
  • Direct plasmid DNA gene transfer is currently the basis of many emerging gene therapy strategies and thus does not require viral genes or lipid particles (Muramatsu et al, 1998; Aihara and Miyazaki, 1998).
  • Skeletal muscle is a preferred target tissue, because muscle fiber has a long life span and can be transduced by circular DNA plasmids that express over months or years in an immunocompetent host (Davis et al, 1993; Tripathy et al, 1996).
  • Wild type GHRH has a relatively short half-life in the circulatory system, both in humans (Frohman et al, 1984) and in farm animals. After 60 minutes of incubation in plasma 95% of the GHRH(1-44)NH2 is degraded, while incubation of the shorter (1-40)OH form of the hormone, under similar conditions, shows only a 77%. degradation of the peptide after 60 minutes of incubation (Frohman et al, 1989).
  • a GHRH analog containing the following mutations has been reported (U.S. Patent No. 5,846,936): Tyr at position 1 to His; Ala at position 2 to Val, Leu, or others; Asn at position 8 to Gin, Ser, or Thr; Gly at position 15 to Ala or Leu; Met at position 27 to Nle or Leu; and Ser at position 28 to Asn.
  • the GHRH analog which is the subject of U.S. Patent Application Serial No. 60/145,624, herein incorporated by reference, does not contain all of the amino acid substitutions reported in U.S. Patent No. 5,846,936 to be necessary for activity.
  • the invention of U.S. Patent Application Serial No. 60/145,624 differs from U.S. Patent No.
  • U.S. Patent Application Serial No. 60/145,624 concerns an analog of growth hormone releasing hormone which differs from the wild type form with significant modifications which improve its function as a GH secretagogue: decreased susceptibility to proteases and increased stability, which would prolong the ability to effect a therapy, and increased biological activity, which would enhance the ability to effect a therapy.
  • the analog of U.S. Patent Application Serial No. 60/145,624 lacks the substitution at position 8 to Gin, Ser, or Thr present in the GHRG analog of U.S. Patent No. 5,756,264.
  • U.S. Patent Application Serial No. 60/145,624 lacks the substitution at position 8 to Gin, Ser, or Thr present in the GHRG analog of U.S. Patent No. 5,756,264.
  • the invention utilizes a DNA encoding the GHRH analog linked to a unique synthetic promoter, termed SPc5-12 (Li et al, 1999), which contains a proximal serum response element (SRE) from skeletal ⁇ -actin, multiple MEF-2 sites, MEF-1 sites, and TEF-1 binding sites, and greatly exceeds the transcriptional potencies of natural myogenic promoters.
  • SPc5-12 Li et al, 1999
  • SRE proximal serum response element
  • the uniqueness of such a synthetic promoter is a significant improvement over, for instance, issued patents concerning a myogenic promoter and its use (e.g. U.S. Pat. No. 5,374,544) or systems for myogenic expression of a nucleic acid sequence (e.g. U.S. Pat. No. 5,298,422).
  • U.S. Patent No. 5,061,690 is directed toward increasing both birth weight and milk production by supplying to pregnant female mammals an effective amount of hGRF or one of its analogs for 10-20 days. Application of the analogs lasts throughout the lactation period. However, multiple administrations are presented, and there is no disclosure regarding administration of the growth hormone releasing hormone (or factor) as a DNA molecule, such as with gene therapy techniques.
  • U.S. Patents No. 5,134,120 and 5,292,721 similarly provide no teachings regarding administration of the growth hormone releasing hormone as a DNA form. Furthermore, these patents concern exclusively multiple administrations of recombinant protein GH in the last 2 weeks of gestation and three weeks after birth. Also, no discussion is provided regarding any non-wild type form, such as is provided in the present invention.
  • GH growth hormone
  • GHRH the upstream stimulator of GH
  • the use of GHRH is an alternate strategy that may increase not only growth performance and milk production, but more importantly, the efficiency of production from both practical and metabolic perspectives (Dubreuil et al, 1990; Farmer et al, 1992).
  • the high cost of the recombinant peptides and the required frequency of administration currently limit the widespread use of this treatment.
  • GHRH Hypothalamic tissue-specific expression ofthe GHRH gene is not required for activity, as extra-cranially secreted GHRH can be biologically active (Faglia et al, 1992; Melmed, 1991).
  • a gene therapy approach to deliver GHRH is favored by the fact that the gene, cDNA and native and several mutated molecules are well characterized in swine, cattle and many other species, and that the determination of therapeutic efficacy is straightforward and unequivocal.
  • the skeletal musculature is a perfect candidate for the target tissue, because intramuscular injection is easily performed in an industrial setting, muscle fibers have a long life span and can be transduced by circular DNA plasmids (Bettan et al, 2000; Everett et al, 2000). Thus, there is no need for re-administration and the transgene can be expressed efficiently over months or years in an immunocompetent host (Wolff et al, 1992).
  • a method of improving or enhancing growth in an offspring from a female animal comprising the step of introducing an effective amount of a vector into cells of the female animal prior to or during gestation of said offspring, wherein the vector comprises a promoter; a nucleotide sequence; and a 3' untranslated region, under conditions wherein the nucleotide sequence is expressed and wherein the introduction and expression ofthe vector results in improved or enhanced growth in the offspring.
  • the cells of said female animal comprise diploid cells.
  • the cells of said female animal comprise muscle cells.
  • the nucleic acid sequence encodes a growth hormone releasing hormone or its analog.
  • the growth hormone releasing hormone is SEQ ID NO:l, SEQ ID NO:8, or its respective analog.
  • the promoter comprises a synthetic myogenic promoter.
  • the 3 ' untranslated region comprises a hGH 3 ' untranslated region.
  • the vector is introduced into said cells of said female animal by electroporation, through a viral vector, in conjunction with a carrier, or by parenteral route.
  • the female animal is a human, a pet animal, a farm animal, a food animal, or a work animal.
  • the female animal is a human, pig, cow, sheep, goat or chicken.
  • the vector is selected from the group consisting of a plasmid, a viral vector, a liposome, and a cationic lipid.
  • the vector is introduced into said female in a single administration.
  • the introduction occurs during the third trimester of gestation of the offspring.
  • the method further comprises the step of administering to the female a ligand for a growth hormone secretagogue receptor.
  • the ligand administration is oral.
  • the present invention there is a method of increasing levels of growth hormone in an offspring from a female animal comprising the step of introducing an effective amount of a vector into cells of the female animal prior to or during gestation of the offspring, wherein the vector comprises a promoter; a nucleotide sequence; and a 3' untranslated region, under conditions wherein the nucleotide sequence is expressed and wherein the introduction and expression of the vector results in an increase in the levels of growth hormone in the offspring.
  • the cells of the female animal comprise diploid cells.
  • the cells ofthe female animal comprise muscle cells.
  • the nucleic acid sequence encodes a growth hormone releasing hormone or its analog.
  • the growth hormone releasing hormone is SEQ ID NO:l, SEQ ID NO:8, or its respective analog.
  • the promoter comprises a synthetic myogenic promoter.
  • the 3 ' untranslated region comprises a hGH 3' untranslated region.
  • the vector is introduced into the cells ofthe female animal by electroporation, through a viral vector, in conjunction with a carrier, or by parenteral route.
  • the female animal is a human, a pet animal, a farm animal, a food animal, or a work animal.
  • the female animal is a human, pig, cow, sheep, goat or chicken.
  • the vector is selected from the group consisting of a plasmid, a viral vector, a liposome, and a cationic lipid.
  • the vector is introduced into the female in a single administration. In an additional specific embodiment, the introduction occurs during the third trimester of gestation of the offspring.
  • the method further comprises the step of administering to the female a ligand for a growth hormone secretagogue receptor. In another specific embodiment, the ligand administration is oral.
  • the present invention there is a method of increasing lean body mass in an offspring from a female animal comprising the step of introducing an effective amount of a vector into cells of the female animal prior to or during gestation of the offspring, wherein the vector comprises a promoter; a nucleotide sequence; and a 3' untranslated region, under conditions wherein the nucleotide sequence is expressed and wherein the introduction and expression of the vector results in increased lean body mass in the offspring.
  • the cells of the female animal comprise diploid cells.
  • the cells of the female animal comprise muscle cells.
  • the nucleic acid sequence encodes a growth hormone releasing hormone or its analog.
  • the growth hormone releasing hormone is SEQ ID NO:l, SEQ ID NO:8, or its respective analog.
  • the promoter comprises a synthetic myogenic promoter.
  • the 3 ' untranslated region comprises a hGH 3 ' untranslated region.
  • the vector is introduced into the cells of the female animal by electroporation, through a viral vector, in conjunction with a carrier, or by parenteral route.
  • the female animal is a human, a pet animal, a farm animal, a food animal, or a work animal.
  • the female animal is a human, pig, cow, sheep, goat or chicken.
  • the vector is selected from the group consisting of a plasmid, a viral vector, a liposome, and a cationic lipid.
  • the vector is introduced into the female in a single administration. In an additional specific embodiment, the introduction occurs during the third trimester of gestation of the offspring.
  • the method further comprises the step of administering to the female a ligand for a growth hormone secretagogue receptor. In another specific embodiment, the ligand administration is oral.
  • a method of increasing levels of IGF-I in an offspring from a female animal comprising the step of introducing an effective amount of a vector into cells of the female animal prior to or during gestation of said offspring, wherein the vector comprises a promoter; a nucleotide sequence; and a 3' untranslated region, under conditions wherein the nucleotide sequence is expressed and wherein said introduction and expression of said vector results in increased levels of IGF-I in the offspring.
  • the cells of the female animal comprise diploid cells.
  • the cells of the female animal comprise muscle cells.
  • the nucleic acid sequence encodes a growth hormone releasing hormone or its analog.
  • the growth hormone releasing hormone is SEQ ID NO:l, SEQ ID NO:8, or its respective analog.
  • the promoter comprises a synthetic myogenic promoter.
  • the 3 ' untranslated region comprises a hGH 3 ' untranslated region.
  • the vector is introduced into the cells of the female animal by electroporation, through a viral vector, in conjunction with a carrier, or by parenteral route.
  • the female animal is a human, a pet animal, a farm animal, a food animal, or a work animal.
  • the female animal is a human, pig, cow, sheep, goat or chicken.
  • the vector is selected from the group consisting of a plasmid, a viral vector, a liposome, and a cationic lipid.
  • the vector is introduced into said female in a single administration.
  • the introduction occurs during the third trimester of gestation of the offspring.
  • the method further comprises the step of administering to the female a ligand for a growth hormone secretagogue receptor.
  • the ligand administration is oral.
  • a method of increasing feed efficiency in an offspring from a female animal comprising the step of introducing an effective amount of a vector into cells of the female animal prior to or during gestation of the offspring, wherein the vector comprises a promoter; a nucleotide sequence; and a 3' untranslated region, under conditions wherein the nucleotide sequence is expressed and wherein the introduction and expression of the vector results in increased feed efficiency in the offspring.
  • the cells of the female animal comprise diploid cells.
  • the cells of the female animal comprise muscle cells.
  • the nucleic acid sequence encodes a growth hormone releasing hormone or its analog.
  • the growth hormone releasing hormone is SEQ ID NO:l, SEQ ID NO:8, or its respective analog.
  • the promoter comprises a synthetic myogenic promoter.
  • the 3' untranslated region comprises a hGH 3' untranslated region.
  • the vector is introduced into the cells of the female animal by electroporation, through a viral vector, in conjunction with a carrier, or by parenteral route.
  • the female animal is a human, a pet animal, a farm animal, a food animal, or a work animal.
  • the female animal is a human, pig, cow, sheep, goat or chicken.
  • the vector is selected from the group consisting of a plasmid, a viral vector, a liposome, and a cationic lipid.
  • the vector is introduced into the female in a single administration. In an additional specific embodiment, the introduction occurs during the third trimester of gestation of the offspring.
  • the method further comprises the step of administering to the female a ligand for a growth hormone secretagogue receptor. In another specific embodiment, the ligand administration is oral.
  • the present invention there is a method of increasing the rate of growth in an offspring from a female animal comprising the step of introducing an effective amount of a vector into cells of the female animal prior to or during gestation of said offspring, wherein the vector comprises a promoter; a nucleotide sequence; and a 3' untranslated region, under conditions wherein the nucleotide sequence is expressed and wherein the introduction and expression of the vector results in increased rate of growth in the offspring.
  • the cells of the female animal comprise diploid cells.
  • the cells of the female animal comprise muscle cells.
  • the nucleic acid sequence encodes a growth hormone releasing hormone or its analog.
  • the growth hormone releasing hormone is SEQ ID NO:l, SEQ ID NO:8, or its respective analog.
  • the promoter comprises a synthetic myogenic promoter.
  • the 3 ' untranslated region comprises a hGH 3 ' untranslated region.
  • the vector is introduced into the cells of said female animal by electroporation, through a viral vector, in conjunction with a carrier, or by parenteral route.
  • the female animal is a human, a pet animal, a farm animal, a food animal, or a work animal.
  • the female animal is a human, pig, cow, sheep, goat or chicken.
  • the vector is selected from the group consisting of a plasmid, a viral vector, a liposome, and a cationic lipid.
  • the vector is introduced into said female in a single administration.
  • the introduction occurs during the third trimester of gestation of the offspring.
  • the method further comprises the step of administering to the female a ligand for a growth hormone secretagogue receptor.
  • the ligand administration is oral.
  • a method of increasing the ratio of somatotrophs to other hormone-producing cells in a pituitary gland of an offspring from a female animal comprising the step of introducing an effective amount of a vector into cells of the female animal prior to or during gestation of the offspring, wherein the vector comprises a promoter; a nucleotide sequence; and a 3' untranslated region, under conditions wherein the nucleotide sequence is expressed and wherein the introduction and expression of the vector results in an increased ratio of somatotrophs to other hormone- producing cells in the offspring.
  • the cells of the female animal comprise diploid cells.
  • the cells of the female animal comprise muscle cells.
  • the nucleic acid sequence encodes a growth hormone releasing hormone or its analog.
  • the growth hormone releasing hormone is SEQ ID NO:l, SEQ ID NO: 8, or its respective analog.
  • the promoter comprises a synthetic myogenic promoter.
  • the 3 ' untranslated region comprises a hGH 3' untranslated region.
  • the vector is introduced into the cells of said female animal by electroporation, through a viral vector, in conjunction with a carrier, or by parenteral route.
  • the female animal is a human, a pet animal, a farm animal, a food animal, or a work animal.
  • the female animal is a human, pig, cow, sheep, goat or chicken.
  • the vector is selected from the group consisting of a plasmid, a viral vector, a liposome, and a cationic lipid.
  • the vector is introduced into the female in a single administration. In an additional specific embodiment, the introduction occurs during the third trimester of gestation of the offspring.
  • the method further comprises the step of administering to the female a ligand for a growth hormone secretagogue receptor.
  • the ligand administration is oral.
  • the hormone-producing cells are selected from the group consisting of corticotrophs, lactotrophs and gonadotrophs.
  • a method for delaying birth of an offspring from a female animal comprising the step of introducing an effective amount of a vector into cells ofthe female animal prior to or during gestation of the offspring, wherein the vector comprises a promoter; a nucleotide sequence; and a 3' untranslated region, under conditions wherein the nucleotide sequence is expressed and wherein the introduction and expression ofthe vector results in delayed birth ofthe offspring.
  • the cells of the female animal comprise diploid cells.
  • the cells of the female animal comprise muscle cells.
  • the nucleic acid sequence encodes a growth hormone releasing hormone or its analog.
  • the growth hormone releasing hormone is SEQ ID NO:l, SEQ ID NO:8, or its respective analog.
  • the promoter comprises a synthetic myogenic promoter.
  • the 3 ' untranslated region comprises a hGH 3 ' untranslated region.
  • the vector is introduced into the cells of said female animal by electroporation, through a viral vector, in conjunction with a carrier, or by parenteral route.
  • the female animal is a human, a pet animal, a farm animal, a food animal, or a work animal.
  • the female animal is a human, pig, cow, sheep, goat or chicken.
  • the vector is selected from the group consisting of a plasmid, a viral vector, a liposome, and a cationic lipid.
  • the vector is introduced into the female in a single administration. In an additional specific embodiment, the introduction occurs during the third trimester of gestation of the offspring.
  • the method further comprises the step of administering to the female a ligand for a growth hormone secretagogue receptor. In another specific embodiment, the ligand administration is oral.
  • a method of increasing milk production in an animal comprising the step of introducing an effective amount of a vector into cells of said animal, wherein said vector comprises a promoter; a nucleotide sequence; and a 3' untranslated region linked, under conditions wherein the nucleotide sequence is expressed and wherein said introduction and expression of said vector results in increased milk production in the animal.
  • the cells of the female animal comprise diploid cells.
  • the cells ofthe female animal comprise muscle cells.
  • the nucleic acid sequence encodes a growth hormone releasing hormone or its analog.
  • the growth hormone releasing hormone is SEQ ID NO: l, SEQ ID NO:8, or its respective analog.
  • the promoter comprises a synthetic myogenic promoter.
  • the 3 ' untranslated region comprises a hGH 3' untranslated region.
  • the vector is introduced into the cells ofthe female animal by electroporation, through a viral vector, in conjunction with a carrier, or by parenteral route.
  • the female animal is a human, a pet animal, a farm animal, a food animal, or a work animal.
  • the female animal is a human, pig, cow, sheep, goat or chicken.
  • the vector is selected from the group consisting of a plasmid, a viral vector, a liposome, and a cationic lipid.
  • the vector is introduced into the female in a single administration. In an additional specific embodiment, the introduction occurs during the third trimester of gestation of the offspring.
  • the method further comprises the step of administering to the female a ligand for a growth hormone secretagogue receptor. In another specific embodiment, the ligand administration is oral.
  • FIGS. 1A through IC demonstrate that GHRH super-active analogs increase GH secretagogue activity and stability.
  • FIG. 1A is a comparison of the porcine wild type (1-40)OH amino acid sequence with the analog HV-GHRH.
  • FIG. IB shows the effect of the different GHRH species on pig GH release in porcine primary pituitary culture.
  • FIG.IC demonstrates changes in stability which occur with HV-GHRH and wild type porcine GHRH during a 6 hour incubation.
  • FIGS. 2 A through 2E demonstrate an increase in GHRH, GH and IGF-I serum levels over two months following single injections of super-active analog GHRH myogenic expression vector.
  • FIG. 2A depicts the constructs which contain the SPc5-12 synthetic promoter and the 3 ' UTR of GH.
  • HV-GHRH construct was used and compared with the porcine wild type as a positive control, and with ⁇ - galactosidase construct as a negative control.
  • FIG. 2B illustrates relative levels of serum GHRH in pSP-GHRH injected pigs versus placebo injected control pigs.
  • FIG. 2C demonstrates absolute levels of serum GHRH in pSP-GHRH injected pigs versus controls pigs corrected for weight/blood volume increase.
  • FIG. 2D shows variation of GH levels in pSP-HV-GHRH injected pigs.
  • FIG. 2E shows plasma IGF-I levels following direct intramuscular injection of pSP-GHRH constructs.
  • FIGS. 3A through 3C demonstrate the effect of myogenic GHRH expression vectors on pig growth.
  • FIG. 3A shows the change in average weight in injected pigs over 2 months with pSP-GHRH or pSP-HV-GHRH.
  • FIG. 3B shows the status of feed conversion efficiency in the pSP-GHRH injected pigs versus controls.
  • FIG. 3C is a comparison of a pSP-HV-GHRH injected pig and a placebo injected control pig, 45 days post-injection.
  • FIG. 4 demonstrates the effect of injection of different amounts of pSP- HV-GHRH on 10 day-old piglets.
  • FIG. 5 shows the effect of injection of different amounts of pSP-HV- GHRH on IGF-I levels in 10 day-old piglets.
  • FIG. 6 illustrates a time course for pSP-HV-GHRH plasmid injection into piglets.
  • FIG. 7 illustrates a preferred embodiment of the present invention for an injectable electrode versus an alternative embodiment of exterior caliper electrodes.
  • On the top is an illustration of external caliper electrodes having 2 square plates/1.5 cm side.
  • On the bottom is an illustration of a 6-needle array device (solid needles) with 18-26 g needles 2cm in length present in a 1cm diameter array.
  • the left illustration is a side view and the right illustration is a bottom view.
  • FIG. 8 demonstrates birth weight of the control and experimental piglets.
  • FIG. 9 illustrates piglet weight at weaning for experimentals and controls.
  • FIG. 10 shows weight of controls cross-fostered to injected animals compared to their littermates.
  • FIG. 11 demonstrates weight of piglets from GHRH-treated sows cross- fostered to control sows and compared to their littermates.
  • FIG. 12 illustrates an overall increase in weight over the controls (fed on controls sows).
  • FIG. 13 shows a comparison of the experimental and control market weights.
  • FIG. 14 illustrates weights of the offspring at 3 weeks, 10 weeks, and 24 weeks.
  • FIG. 15 shows muscle weight per body weight at three weeks of age.
  • FIG. 16 demonstrates pituitary weight per total weight ofthe offspring.
  • FIG. 17 shows RNA analysis of GH, GHRH, and PRL in the offspring, illustrating GHRH acts in utero as a growth factor on the pituitary.
  • FIG. 18 illustrates DAB staining of GH-secreting cells.
  • FIG. 19 demonstrates IGF-I concentration in offspring at 3 weeks, 12 weeks, and 6 months.
  • animal refers to any species of the animal kingdom. In preferred embodiments it refers more specifically to humans, animals in their wild state, animals used as pets (birds, dogs, cats, horses), animals used for work (horses, cows, dogs) and animals which produce food (chickens, cows, fish), farm animals (pigs, horses, cows, sheep, chickens) or are themselves food (frogs, chickens, fish, crabs, lobsters, shrimp, mussels, scallops, goats, boars, cows, lambs, pigs, ostrich, emu, eel) and other animals well known to the art.
  • the term "effective amount” as used herein is defined as the amount ofthe composition required to produce an effect in a host which can be monitored using several endpoints known to those skilled in the art. In a specific embodiment, these endpoints are surrogate markers.
  • feed conversion efficiency is defined as the amount of food an animal eats per day versus the amount of weight gained by said animal.
  • efficiency or feed efficiency as used herein is interchangeable with “feed conversion efficiency.”
  • growth deficiencies is defined as any health status, medical condition or disease in which growth is less than normal.
  • the deficiency could be the result of an aberration directly affecting a growth hormone pathway (such as the GHRH-GH-IGF-I axis), indirectly affecting a growth hormone pathway, or not affecting a growth hormone pathway at all.
  • growth hormone as used herein is defined as a hormone which relates to growth and acts as a chemical messenger to exert its action on a target cell.
  • growth hormone releasing hormone as used herein is defined as a hormone which facilitates or stimulates release of growth hormone.
  • growth hormone releasing hormone analog as used herein is defined as a protein which contains amino acid mutations and/or deletions in the naturally occurring form of the amino acid sequence (with no synthetic dextro or cyclic amino acids), but not naturally occurring in the GHRH molecule, yet still retains its function to enhance synthesis and secretion of growth hormone.
  • growth hormone secretagogue receptor as used herein is defined as a receptor for a small synthetic compound which is associated, either directly or indirectly, with release of growth hormone from the pituitary gland.
  • lean body mass is defined as the mass of the body of an animal attributed to non-fat tissue, such as muscle.
  • ligand for a growth hormone secretagogue receptor is defined as any compound which acts as an agonist on a growth hormone secretagogue receptor.
  • the ligand may be synthetic or naturally occurring.
  • the ligand may be a peptide, protein, sugar, carbohydrate, lipid, nucleic acid or a combination thereof.
  • muscle tissue refers specifically to muscle tissue.
  • newborn refers to an animal immediately after birth and all subsequent stages of maturity or growth.
  • offspring refers to a progeny of a parent, wherein the progeny is an unborn fetus or a newborn.
  • parenteral refers to a mechanism for introduction of material into an animal other than through the intestinal canal.
  • parenteral includes subcutaneous, intramuscular, intravenous, intrathecal, intraperitoneal, or others.
  • pharmaceutically acceptable refers to a compound wherein administration of said compound can be tolerated by a recipient mammal.
  • secretagogue refers to a natural of synthetic molecule that enhances synthesis and secretion of a downstream - regulated molecule (e.g. GHRH is a secretagogue for GH).
  • somatotroph refers to a cell which produces growth hormone.
  • terapéuticaally effective amount refers to the amount of a compound administered wherein said amount is physiologically significant.
  • An agent is physiologically significant if its presence results in technical change in the physiology of a recipient animal. For example, in the treatment of growth deficiencies, a composition which increases growth would be therapeutically effective; in consumption diseases a composition which would decrease the rate of loss or increase the growth would be therapeutically effective.
  • the term "vector” as used herein refers to any vehicle which delivers a nucleic acid into a cell or organism. Examples include plasmids, viral vectors, liposomes, or cationic lipids. In a specific embodiment, liposomes and cationic lipids are adjuvant (carriers) that can be complexed with other vectors to increase the uptake of plasmid or viral vectors by a target cell.
  • the vector comprises a promoter, a nucleotide sequence, preferably encoding a growth hormone releasing hormone or its analog, and a 3' untranslated region. In another preferred embodiment, the promoter, nucleotide sequence, and 3 ' untranslated region are linked operably for expression in a eukaryotic cell.
  • wasting symptoms as used herein is defined as symptoms and conditions associated with consumption or chronic wasting diseases.
  • This application is related in subject matter to U.S. Provisional Patent Application No. 60/145,624, filed July 26, 1999 and the corresponding U.S. Nonprovisional Patent Application No. 09/624,268 filed July 24, 2000, both herein incorporated by reference.
  • GHRH growth hormone releasing hormone
  • piglets bom sows treated with a gene therapy using a plasmid DNA constructs encoding for GHRH show an increase in growth pattern over normal levels to at least 170 days after birth, and are leaner, while maintaining a normal homeostasis. This increase is equally due to increase milk production in the injected sows and modification of the hypothalamic - pituitary axis in the offspring.
  • This proof of principal experiment demonstrate that plasmid mediated transfer could be used to enhance certain animal characteristics throughout generations, while avoiding secondary effects linked with classical protein treatments.
  • a nucleic acid sequence is utilized in the methods of the present invention which increases growth, enhances growth, increases feed conversion efficiency, increases lean body mass, increases IGF-I levels, increases growth rate, increases the ratio of somatotrophs to other hormone-producing cells, delays birth, or increases milk production in an offspring of a female.
  • the nucleic acid sequence is growth hormone releasing hormone, growth hormone, IGF-I, prolactin, or analogs thereof.
  • the female may be a mother, a female who has never been pregnant or given birth before, or a surrogate mother, such as impregnated by fetal transplantation.
  • a preferred embodiment of the present invention utilizes the growth hormone-releasing hormone analog having the amino acid sequence of SEQ ID NO:l or SEQ ID NO:8 (wt GHRH).
  • wild-type can be the endogenous form of GHRH of any animal, or it may be a slightly modified form of the hormone, such as the porcine GHRH.
  • porcine GHRH a naturally modified form of the hormone
  • the endogenous GHRH has 44 amino acids, and an amide group at the end, with the correct notation for that form being (1-44)NH 2 - GHRH.
  • a form with only 40 amino acids (lacking the last 4 amino acids) which also does not contain an amide group, and may be referred to as (1- 40)OH-GHRH.
  • This form as used herein may also be referred to as wild-type because it does not contain internal mutations if compared to the wild-type sequence, as opposed to other forms discussed herein (such as the HV) having internal mutations introduced by site- directed mutagenesis.
  • the 1-40 form and shorter forms exist naturally in humans and other mammals (even in different types of GHRH secreting tumors), and they have an activity comparable with the natural (1- 44)NH 2 .
  • a GHRH with increased stability over wild type GHRH is utilized.
  • U.S. Patent No. 4,223,019 discloses pentapeptides having the amino acid sequence NH 2 — Y--Z--E- -G--J — COOH, wherein Y is selected from a group consisting of D-lysine and D-arginine; Z and J are independently selected from a group consisting of tyrosine, tryptophan, and phenylalanine; and E and G are independently selected from a group consisting of D-tyrosine, D-tryptophan, and D-phenylalanine.
  • 4,223,020 discloses tetrapeptides having the following amino acid sequence NH 2 ⁇ Y ⁇ Z— E--G--COOH wherein Y and G are independently selected from a group consisting of tyrosine, tryptophan, and phenylalanine; and Z and E are independently selected from a group consisting of D-tyrosine, D-tryptophan, and D-phenylalanine.
  • Y and G are independently selected from a group consisting of tyrosine, tryptophan, and phenylalanine
  • Z and E are independently selected from a group consisting of D-tyrosine, D-tryptophan, and D-phenylalanine.
  • 4,223,021 discloses pentapeptides having the following amino acid sequence NH 2 -Y--Z— E-G-J--COOH wherein Y and G are independently selected from a group consisting of tyrosine, tryptophan, and phenylalanine; Z is selected from a group consisting of glycine, alanine, valine, leucine, isoleucine, proline, hydroxyproline, serine, threonine, cysteine, and methionine; and E and J are independently selected from a group consisting of D-tyrosine, D-tryptophan, and D-phenylalanine.
  • 4,224,316 discloses novel pentapeptides having the following amino acid sequence NH 2 -Y-Z-E-G-J-COOH wherein Y and E are independently selected from a group consisting of D-tyrosine, D-tryptophan, and D-phenylalanine; Z and G are independently selected from a group consisting of tyrosine, tryptophan, and phenylalanine; and J is selected from a group consisting of glycine, alanine, valine, leucine, isoleucine, proline, hydroxyproline, serine, threonine, cysteine, methionine, aspartic acid, glutamic acid, asparagine, glutamine, arginine, and lysine.
  • U.S. Patent No. 4,226,857 discloses pentapeptides having the following amino acid sequence NH 2 -Y-Z-E-G-J-COOH wherein Y and G are independently selected from a group consisting of tyrosine, trytophan, and phenylalanine; Z and J are independently selected from a group consisting of D-tyrosine, D- tryptophan, and D-phenylalanine; and E is selected from a group consisting of glycine, alanine, valine, leucine, isoleucine, proline, hydroxyproline, serine, threonine, cysteine, methionine, aspartic acid, glutamic acid, asparagine, glutamine, and histidine.
  • U.S. Patent No. 4,228,155 discloses pentapeptides having the following amino acid sequence NH 2 -Y-Z- E-G-J-COOH wherein Y is selected from a group consisting of tyrosine, D-tyrosine, tryptophan, D-tryptophan, phenylalanine, and D-phenylalanine; Z and E are independently selected from a group consisting of D-tyrosine, D-tryptophan, and D-phenylalanine; G is selected from a group consisting of lysine and arginine; and J is selected from a group consisting of glycine, alanine, valine, leucine, isoleucine, proline, hydroxyproline, serine, threonine, cysteine, and methionine.
  • U.S. Patent No. 4,228,156 discloses tripeptides having the following amino acid sequence NH 2 -Y-Z-E-COOH wherein Y and Z are independently selected from a group consisting of D-tyrosine, D-tryptophan, and D-phenylalanine; and E is selected from a group consisting of tyrosine, tryptohan, and phenylalanine.
  • Y and Z are independently selected from a group consisting of D-tyrosine, D-tryptophan, and D-phenylalanine
  • E is selected from a group consisting of tyrosine, tryptohan, and phenylalanine.
  • 4,228,158 discloses pentapeptides having the following amino acid sequence NH 2 — Y--Z-- E ⁇ G--J--COOH wherein Y and G are independently selected from a group consisting of tyrosine, tryptophan, and phenylalanine, Z and E are independently selected from a group consisting of D-tyrosine, D-tryptophan, and D-phenylalanine; and J is selected from a group consisting of natural amino acids and the D-configuration thereof.
  • 4,833,166 discloses a synthetic peptide having the formula: H-Asp-Pro-Val-Asn-Ile-Arg-Ala-Phe-Asp- Asp-Val-Leu-Y wherein Y is OH or NH 2 or a non-toxic salt thereof and A synthetic peptide having the formula: H-Val-Glu-Pro-Gly-Ser-Leu-Phe-Leu-Val-Pro-Leu-Pro-Leu-Leu-Pro- Val-His-Asp-Phe-Val-Gln-Gln-Phe-Ala-Gly-Ile-Y wherein Y is OH or NH 2 or a non-toxic salt thereof.
  • Draghia-Akli et al (1997) utilize a 228-bp fragment of hGHRH which encodes a 31-amino-acid signal peptide and an entire mature peptide human GHRH(l-44)OH (Tyrl Leu44) originally described by Mayo et al. (1995).
  • Guillemin et al. (1982) also determine the sequence of human pancreatic growth hormone releasing factor (hpGRF).
  • Additional embodiments of the present invention include: (1) a method for improving growth performance in an offspring; (2) a method for stimulating production of growth hormone in an offspring at a level greater than that associated with normal growth; and (3) a method of enhancing growth in an offspring. All of these methods include the step of introducing a plasmid vector into the mother of the offspring during gestation of the offspring or during a previous pregnancy, wherein said vector comprises a promoter; a nucleotide sequence, such as one encoding SEQ ID NO:l or SEQ ID NO:8; and a 3' untranslated region operatively linked sequentially at appropriate distances for functional expression.
  • a method for stimulating production of growth hormone in an offspring at a level greater than that associated with normal growth comprising introducing into the mother of said offspring during the gestation of said offspring an effective amount of a vector, said vector comprising a promoter; a nucleotide sequence encoding SEQ ID NO:l or SEQ ID NO:8; and a 3' untranslated region operatively linked sequentially at appropriate distances for functional expression.
  • a level greater than that associated with normal growth includes the basal, inherent growth of an animal with a growth-related deficiency or of an animal with growth levels similar to other similar animals in the population, including those with no growth- related deficiency.
  • a method of enhancing growth in an animal comprising introducing into said animal an effective amount of a vector, said vector comprising a promoter; a nucleotide sequence encoding SEQ ID NO:l or SEQ ID NO:8; and a 3' untranslated region operatively linked sequentially at appropriate distances for functional expression.
  • the animal whose growth is enhanced may or may not have a growth deficiency.
  • the growth and/or growth rate of an animal is affected for long terms, such as greater than a few weeks or greater than a few months. In a specific embodiment, this is achieved by administering growth hormone releasing hormone into the mother ofthe offspring, preferably in a nucleic acid form.
  • the GHRH nucleic acid is maintained as an episome in a muscle cell.
  • the increase in GHRH affects the pituitary gland by increasing the number of growth hormone producing cells, and thus changes their cellular lineage.
  • the ratio of somatotrophs is increased relative to other hormone producing cells in the pituitary, such as corticotrophs, lactotrophs, gonadotrophs, etc.
  • the increase in growth hormone related to the increase in the number of growth hormone- producing cells, is reflected in an increase of IGF-I levels.
  • the increase in growth hormone levels is associated with an increase in lean body mass and an increase in the rate of growth of the offspring.
  • the increase in lean body mass is related to the increase in linear skeletal growth.
  • the feed conversion efficiency of the offspring is increased.
  • birth of the offspring is delayed, and in a preferred embodiment this is associated with an improved or increased growth rate ofthe fetus.
  • the promoter is a synthetic myogenic promoter and hGH 3' untranslated region is in the 3' untranslated region.
  • the 3' untranslated region may be from any natural or synthetic gene.
  • a synthetic promoter termed SPc5-12 (Li et al, 1999) (SEQ ID NO:6), which contains a proximal serum response element (SRE) from skeletal ⁇ -actin, multiple MEF-2 sites, MEF-1 sites, and TEF-1 binding sites, and greatly exceeds the transcriptional potencies of natural myogenic promoters.
  • the promoter utilized in the invention does not get shut off or reduced in activity significantly by endogenous cellular machinery or factors.
  • tr ⁇ «5-acting factor binding sites and enhancers may be used in accordance with this embodiment of the invention.
  • a natural myogenic promoter is utilized, and a skilled artisan is aware how to obtain such promoter sequences from databases including the National Center for Biotechnology Information (NCBI) GenBank database or the NCBI PubMed site. A skilled artisan is aware that these World Wide Web sites may be utilized to obtain sequences or relevant literature related to the present invention.
  • NCBI National Center for Biotechnology Information
  • the hGH 3' untranslated region (SEQ ID NO: 7) is utilized in a nucleic acid vector, such as a plasmid.
  • said vector is selected from the group consisting of a plasmid, a viral vector, a liposome, or a cationic lipid.
  • said vector is introduced into myogenic cells or muscle tissue.
  • said animal is a human, a pet animal, a work animal, or a food animal.
  • a targeted system for non-viral forms of DNA or RNA requires four components: 1) the DNA or RNA of interest; 2) a moiety that recognizes and binds to a cell surface receptor or antigen; 3) a DNA binding moiety; and 4) a lytic moiety that enables the transport of the complex from the cell surface to the cytoplasm.
  • liposomes and cationic lipids can be used to deliver the therapeutic gene combinations to achieve the same effect.
  • Potential viral vectors include expression vectors derived from viruses such as adenovirus, vaccinia virus, herpes virus, and bovine papilloma virus. In addition, episomal vectors may be employed. Other DNA vectors and transporter systems are known in the art.
  • expression vectors derived from various bacterial plasmids, retroviruses, adenovirus, herpes or from vaccinia viruses may be used for delivery of nucleotide sequences to a targeted organ, tissue or cell population. Methods which are well known to those skilled in the art can be used to construct recombinant vectors which will express the gene encoding the growth hormone releasing hormone analog. Transient expression may last for a month or more with a non-replicating vector and even longer if appropriate replication elements are a part ofthe vector system.
  • a single administration of a growth hormone releasing hormone is sufficient for multiple gestation periods and also provides a therapy that enhances piglets performances to the market weight, as increased growth and changed body composition.
  • vector is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where the vector can be replicated and the nucleic acid sequence can be expressed.
  • a nucleic acid sequence can be "exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found.
  • Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
  • YACs artificial chromosomes
  • expression vector refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed.
  • the nucleic acid sequence encodes part or all of GHRH.
  • RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes.
  • Expression vectors can contain a variety of "control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.
  • the vector ofthe present invention is a plasmid which comprises a synthetic myogenic (muscle-specific) promoter, a nucleotide sequence encoding a growth hormone releasing hormone or its analog, and a 3 ' untranslated region.
  • the vectors is a viral vector, such as an adeno-associated virus, an adenovirus, or a retrovirus.
  • skeletal alpha-actin promoter, myosin light chain promoter, cytomegalovirus promoter, or SV40 promoter can be used.
  • human growth hormone, bovine growth hormone, SV40, or skeletal alpha actin 3 ' untranslated regions are utilized in the vector, a. Promoters and Enhancers
  • a “promoter” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors.
  • the phrases "operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence.
  • a promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
  • a promoter may be one of naturally-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous.”
  • an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
  • certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.
  • a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment.
  • promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCRTM, in connection with the compositions disclosed herein (see U.S. Patent 4,683,202, U.S. Patent 5,928,906, each incorporated herein by reference).
  • control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
  • promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression.
  • Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et ,al (1989), incorporated herein by reference.
  • the promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides.
  • the promoter may be heterologous or endogenous.
  • the promoter is a synthetic myogenic promoter, such as is described in Li et al. (1999).
  • tissue-specific promoters or elements as well as assays to characterize their activity, is well known to those of skill in the art.
  • regions include the human LIMK2 gene (Nomoto et al. 1999), the somatostatin receptor 2 gene (Kraus et al, 1998), murine epididymal retinoic acid-binding gene (Lareyre et al, 1999), human CD4 (Zhao-Emonet et al, 1998), mouse alpha2 (XI) collagen (Tsumaki, et al, 1998), D1A dopamine receptor gene (Lee, et al, 1997), insulin-like growth factor II (Wu et al, 1997), human platelet endothehal cell adhesion molecule-1 (Almendro et al, 1996).
  • a specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.
  • IRES elements are used to create multigene, or polycistronic, messages.
  • IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988).
  • IRES elements from two members of the picornavirus family polio and encephalomyocarditis have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Samow, 1991).
  • IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages.
  • each open reading frame is accessible to ribosomes for efficient translation.
  • Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Patent 5,925,565 and 5,935,819, herein incorporated by reference).
  • Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector.
  • MCS multiple cloning site
  • Restriction enzyme digestion refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art.
  • a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector.
  • "Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology. d. Splicing Sites
  • RNA molecules will undergo RNA splicing to remove introns from the primary transcripts.
  • Vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing ofthe transcript for protein expression. (See Chandler et al, 1997, herein incorporated by reference.) e. Polyadenylation Signals
  • polyadenylation signal to effect proper polyadenylation of the transcript.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed.
  • Preferred embodiments include the SV40 polyadenylation signal and/or the bovine or human growth hormone polyadenylation signal, convenient and/or known to function well in various target cells.
  • Also contemplated as an element of the expression cassette is a transcriptional termination site. These elements can serve to enhance message levels and/or to minimize read through from the cassette into other sequences. f. Origins of Replication
  • a vector in a host cell may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated.
  • ori origins of replication sites
  • ARS autonomously replicating sequence
  • the cells contain nucleic acid construct of the present invention
  • a cell may be identified in vitro or in vivo by including a marker in the expression vector.
  • markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector.
  • a selectable marker is one that confers a property that allows for selection.
  • a positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection.
  • An example of a positive selectable marker is a drug resistance marker.
  • a drug selection marker aids in the cloning and identification of transformants
  • genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
  • markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated.
  • screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized.
  • the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.
  • "host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector.
  • a host cell can, and has been, used as a recipient for vectors.
  • a host cell may be "transfected” or “transformed,” which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a transformed cell includes the primary subject cell and its progeny.
  • Host cells may be derived from prokaryotes or eukaryotes, depending upon whether the desired result is replication of the vector or expression of part or all of the vector-encoded nucleic acid sequences.
  • Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org).
  • ATCC American Type Culture Collection
  • An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result.
  • a plasmid or cosmid for example, can be introduced into a prokaryote host cell for replication of many vectors.
  • Bacterial cells used as host cells for vector replication and/or expression include DH5a, JM109, and KC8, as well as a number of commercially available bacterial hosts such as SURE ® Competent Cells and SOLOPACKa Gold Cells (STRATAGENE ® , La Jolla). Altematively, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses.
  • Examples of eukaryotic host cells for replication and/or expression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.
  • Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.
  • compositions discussed above Numerous expression systems exist that comprise at least a part or all of the compositions discussed above.
  • Prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.
  • the insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patent No. 5,871,986, 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC ® 2.0 from INVITROGEN ® and BACPACKTM BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH ® .
  • mutagenesis will be accomplished by a variety of standard, mutagenic procedures. Mutation is the process whereby changes occur in the quantity or structure of an organism. Mutation can involve modification of the nucleotide sequence of a single gene, blocks of genes or whole chromosome. Changes in single genes may be the consequence of point mutations which involve the removal, addition or substitution of a single nucleotide base within a DNA sequence, or they may be the consequence of changes involving the insertion or deletion of large numbers of nucleotides.
  • Mutations can arise spontaneously as a result of events such as errors in the fidelity of DNA replication or the movement of transposable genetic elements (transposons) within the genome. They also are induced following exposure to chemical or physical mutagens.
  • mutation-inducing agents include ionizing radiations, ultraviolet light and a diverse array of chemical such as alkylating agents and polycyclic aromatic hydrocarbons all of which are capable of interacting either directly or indirectly (generally following some metabolic biotransformations) with nucleic acids.
  • the DNA lesions induced by such environmental agents may lead to modifications of base sequence when the affected DNA is replicated or repaired and thus to a mutation. Mutation also can be site-directed through the use of particular targeting methods.
  • Structure-guided site-specific mutagenesis represents a powerful tool for the dissection and engineering of protein- ligand interactions (Wells, 1996, Braisted et al, 1996).
  • the technique provides for the preparation and testing of sequence variants by introducing one or more nucleotide sequence changes into a selected DNA.
  • Site-specific mutagenesis uses specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent, unmodified nucleotides. In this way, a primer sequence is provided with sufficient size and complexity to form a stable duplex on both sides of the deletion junction being traversed. A primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction ofthe sequence being altered.
  • the technique typically employs a bacteriophage vector that exists in both a single-stranded and double-stranded form.
  • Vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double-stranded plasmids are also routinely employed in site-directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.
  • An oligonucleotide primer bearing the desired mutated sequence, synthetically prepared, is then annealed with the single-stranded DNA preparation, taking into account the degree of mismatch when selecting hybridization conditions.
  • the hybridized product is subjected to DNA polymerizing enzymes such as E. coli polymerase I (Klenow fragment) in order to complete the synthesis of the mutation- bearing strand.
  • E. coli polymerase I Klenow fragment
  • compositions comprising a promoter; a nucleotide sequence encoding SEQ ID NO:l or SEQ ID NO: 8; and a 3' untranslated region operatively linked sequentially at appropriate distances for functional expression
  • compositions can be formulated and administered to affect a variety of growth deficiency states by any means that produces contact of the active ingredient with the agent's site of action in the body of an animal.
  • composition of the present invention is defined as a vector containing a nucleotide sequence encoding the compound of the invention, which is an amino acid sequence analog herein described. Said composition is administered in sufficient quantity to generate a therapeutically effective amount of said compound.
  • administered and “introduced” can be used interchangeably. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. In a preferred embodiment the active ingredient is administered alone or in a buffer such as PBS, but may be administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • Such pharmaceutical compositions can be used for therapeutic or diagnostic purposes in clinical medicine, both human and veterinary.
  • they are useful in the treatment of growth-related disorders such as hypopituitary dwarfism resulting from abnormalities in growth hormone production. Furthermore they can also be used to stimulate the growth or enhance feed conversion efficiency of animals raised for meat production, to enhance milk production, and stimulate egg production.
  • the dosage administered will be a therapeutically effective amount of active ingredient and will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular active ingredient and its mode and route of administration; type of animal; age of the recipient; sex of the recipient; reproductive status of the recipient; health of the recipient; weight of the recipient; nature and extent of symptoms; kind of concurrent treatment; frequency of treatment; and the effect desired.
  • Appropriate dosages of the vectors of the invention to be administered will vary somewhat depending on the individual subject and other parameters. The skilled practitioner will be able to determine appropriate dosages based on the known circulating levels of growth hormone associated with normal growth and the growth hormone releasing activity of the vector.
  • a method of increasing growth of an offspring which comprises administering to the female or mother of the offspring an amount ofthe analog of this invention sufficient to increase the production of growth hormone to levels greater than that which is associated with normal growth. Normal levels of growth hormone vary considerably among individuals and, for any given individual, levels of circulating growth hormone vary considerably during the course of a day.
  • the gene therapy vectors can be formulated into preparations in solid, semisolid, liquid or gaseous forms in the ways known in the art for their respective route of administration. Means known in the art can be utilized to prevent release and absorption of the composition until it reaches the target organ or to ensure timed-release of the composition.
  • a pharmaceutically acceptable form should be employed which does not ineffectuate the compositions of the present invention.
  • the compositions can be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
  • the pharmaceutical composition ofthe present invention may be delivered via various routes and to various sites in an animal body to achieve a particular effect (see, e.g., Rosenfeld et al. (1991); Rosenfeld et al, (1991a); Jaffe et al, 1992).
  • a particular route can provide a more immediate and more effective reaction than another route.
  • Local or systemic delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration.
  • a vector into a cell examples include: (1) methods utilizing physical means, such as electroporation (electricity), a gene gun (physical force) or applying large volumes of a liquid (pressure); and (2) methods wherein said vector is complexed to another entity, such as a liposome or transporter molecule.
  • the present invention provides a method of transferring a therapeutic gene to a host, which comprises administering the vector of the present invention, preferably as part of a composition, using any of the aforementioned routes of administration or alternative routes known to those skilled in the art and appropriate for a particular application.
  • Effective gene transfer of a vector to a host cell in accordance with the present invention to a host cell can be monitored in terms of a therapeutic effect (e.g. alleviation of some symptom associated with the particular disease being treated) or, further, by evidence of the transferred gene or expression of the gene within the host (e.g.
  • compositions can be further approximated through analogy to compounds known to exert the desired effect.
  • the actual dose and schedule can vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on interindividual differences in pharmacokinetics, drug disposition, and metabolism.
  • amounts can vary in in vitro applications depending on the particular cell line utilized (e.g., based on the number of vector receptors present on the cell surface, or the ability of the particular vector employed for gene transfer to replicate in that cell line).
  • the amount of vector to be added per cell will likely vary with the length and stability of the therapeutic gene inserted in the vector, as well as the nature of the sequence, and is particularly a parameter which needs to be determined empirically, and can be altered due to factors not inherent to the methods of the present invention (for instance, the cost associated with synthesis).
  • One skilled in the art can easily make any necessary adjustments in accordance with the exigencies ofthe particular situation.
  • GHRH has a relatively short half-life of about 12 minutes in the circulatory systems of both humans (Frohman et al, 1984) and pigs.
  • GHRH analogs that prolong its biological half-life and/or improve its GH secretagogue activity, enhanced GH secretion is achieved.
  • GHRH mutants were generated by site directed mutagenesis. Gly 15 was substituted for Alal5 to increase ⁇ -helical conformation and amphiphilic structure to decrease cleavage by trypsin-like enzymes (Su et al, 1991).
  • GHRH analogs with Alal5 substitutions display a 4-5 fold greater affinity for the GHRH receptor
  • Dipeptidyl peptidase IV is the prime serum GHRH degradative enzyme (Walter et al, 1980; Martin et al, 1993). Poorer dipeptidase substrates were created by taking GHRH15/27/28 and then by replacing Ile2 with Ala2 (GHRH-TI) or with Val2 (GHRH-TV), or by converting Tyrl and
  • a plasmid of SEQ ID NO:9 (pSPc5-12-HV- GHRH is utilized in the present invention.
  • a plasmid vector is utilized wherein the plasmid comprises a pVC0289 backbone (SEQ ID NO: 10); a promoter, such as of SEQ ID NO:6; a GHRH cDNA, such as the porcine HV-GHRH (the mutated HV-GHRH cDNA) (SEQ ID NO:l 1); and a 3' UTR, such as from human GH (SEQ ID NO:7).
  • plasmid vectors were engineered that were capable of directing the highest level of skeletal muscle-specific gene expression by a newly described synthetic muscle promoter, SPc5-12, which contains a proximal serum response element from skeletal ⁇ -actin, multiple MEF-2 sites, multiple MEF-1 sites, and TEF-1 binding sites (Li et al, 1999).
  • a 228-bp fragment of porcine GHRH which encodes the 31 amino acid signal peptide and the entire mature peptide porcine GHRH (Tyrl-Gly40) and or the GHRH mutants, followed by the 3' untranslated region of human GH cDNA, were incorporated into myogenic GHRH expression vectors by methods well known in the art.
  • the plasmid pSPc5-12 contains a
  • the human GHRH cDNA was subcloned as a BamHI-Hind III fragment into the corresponding sites of the pALTER Promega vector and mutagenesis was performed according to the manufacturer's directions.
  • the porcine wild type cDNA was obtained from the human cDNA by changing the human amino acids 34 and
  • the porcine HV mutations were made with the primer of SEQ ID NO:3:
  • pituitary tissue was dissociated under enzymatic conditions, plated on plastic dishes for enough time to allow attachment. The cells were then rinsed and exposed to incubation media prior to experiments. For details see Tanner et al (1990).
  • Endotoxin-free plasmid (Qiagen Inc., Chatsworth, CA) preparations of pSPc5-12-HV-GHRH, pSPc5-12-wt-GHRH and pSPc5-12bgal were diluted in PBS (pH 7.4) to lmg/ml. The animals were assigned equally to one of the treatments. The pigs were anesthetized with isoflurane (concentration of 2-6%> for induction and l-3%> for maintenance). Jugular catheters were implanted by surgical procedure to draw blood from the animals at day 3, 7, 14, 21, 28, 45 and 65 post-injection.
  • lOmg of plasmid was injected directly into the semitendinosus muscle of pigs. Two minutes after injection, the injected muscle was placed in between a set of calipers and electroporated using optimized conditions of 200V/cm with 4 pulses of 60 milliseconds (Aihara et al, 1998). At 65 days post-injection, animals were killed and internal organs and injected muscle collected, weighed, frozen in liquid nitrogen, and stored at -80°C. Carcasses were weighed and analyzed by neutron activation. Back fat was measured.
  • FIG.2A Three-week-old castrated male-pigs were anesthetized and a jugular vein catheter was inserted to allow collection of blood samples with no discomfort for the animals. Plasmid expression vector
  • DNA (10 mg of DNA of pSP-HV-GHRH; pSP-wt-GHRH; or pSP- ⁇ gal) was injected directly into semitendinosus muscle, which was then electroporated (See Example 7) .
  • Porcine GHRH was measured by a heterologous human assay system (Peninsula Laboratories, Belmont, CA). Sensitivity of the assay is 1 pg/tube. Porcine GH in plasma was measured with a specific double antibody procedure RIA (The Pennsylvania State University). The sensitivity of the assay is 4ng/tube. Porcine IGF-I was measured by heterologous human assay (Diagnostic System Lab., Webster, TX). Data are analyzed using Microsoft Excel statistics analysis package. Values shown in the figures are the mean ⁇ s.e.m. Specific p values were obtained by comparison using Students t test. A p ⁇ 0.05 is set as the level of statistical significance.
  • GHRH levels was increased at 7 days post-injection (FIG.2B), and were 150% above the control levels at 14 days (652.4 ⁇ 77pg/ml versus 419.6 ⁇ 13pg/ml).
  • pSP-HV-GHRH expression activity reached a plateau by 60 days that was about 2 to 3 fold greater levels than the placebo injected control values.
  • FIG.2E Another indication of increased systemic levels of * GH would be elevated levels of IGF-I.
  • Serum porcine IGF-I levels started to rise in pSP-HV-GHRH injected pigs at about 3 days post-injection (FIG.2E). At 21 days, these animals averaged about a 3-fold increase in serum IGF-I levels, which was maintained over 60 days (p ⁇ 0.03).
  • Feed conversion efficiency was also improved by 20%> in pigs injected with GHRH constructs when compared with controls (0.267 kg of food/day for each kg weight gain in pSP-HV-GHRH, and 0.274 kg in pSP-wt-GHRH, versus 0.334 kg in pSP- ⁇ gal injected pigs (FIG.3B).
  • Body composition studies by densitometry, K40 potassium chamber and neutron activation chamber showed a proportional increase of all body components in GHRH injected animals, with no signs of organomegaly, relative proportion of body fat and associated pathology.
  • a photograph of a placebo injected control pig and a pSP-HV-GHRH injected pig after 45 days is shown in FIG.3C.
  • Serum glucose level was similar between the controls and the plasmid GHRH injected pigs (99.2 ⁇ 4.8mg/dl in control pigs, 104.8 ⁇ 6.9mg/dl in pSP-HV-GHRH injected pigs and 97.5 ⁇ 8mg/dl in wild-type pSP- GHRH injected animals (p ⁇ 0.27). No other metabolic changes were found.
  • mice were injected with the indicated doses of pSP- HV-GHRH 10 days after birth.
  • IGF-I values started to rise 10 days post-injection, and at 35 days post-injection pigs injected with 100 micrograms plasmid averaged 10.62 fold higher IGF-I than the controls. Pigs injected with 1 mg averaged 7.94 fold over the controls, and pigs injected with 3 mg averaged 1.16 fold over control values.
  • lower dosages of pSP-HV-GHRH are injected.
  • about 100 micrograms (.1 milligrams) of the plasmid is utilized.
  • about 200-300 micrograms are injected.
  • 50-100 micrograms are administered.
  • IGF-I levels are the lowest (about 10-14 days of life), and may be counterproductive when
  • GHRH levels are normally high.
  • an optimal time point for injection is 14 days after birth (an average 8 pounds heavier than the controls (p ⁇ 0.04) at 40 days post-injection).
  • a preferred dosage for injection is 100 micrograms plasmid in 2-5 ml volume (an average 6 pounds heavier than the controls (p ⁇ 0.02) at 40 days post-injection).
  • Hormonal and biochemical constants are normal (IGF-I, IGF-BP3, insulin, urea, glucose, total proteins, creatinine) in the offspring of sow 1 (time course) and sow 3 (dose curve) and in correlation with weight increase, with no deleterious side effects.
  • Body composition studies from the previous experiment showed that HV-GHRH determined a uniform increase of all body compartments (body composition similar to the controls but bigger), while wt-GHRH determined an increase in lean body mass and a decrease in fat.
  • the sow ( ⁇ 800 pounds) was injected with 10 mg of a pSP-HV-GHRH vector at 90 days of gestation in her first pregnancy. Delivery methods may be any known in the art.
  • the plasmid is delivered as in Example 7 with the exception that a caliper electrode for electroporation was utilized (FIG.7).
  • the electrode has six needles 22g which are 2 cm in length and which are on a circular plastic support of 1 cm in diameter.
  • Table 2 demonstrates the weight (kg) over time of piglets bom from a sow injected with pSP-HV-GHRH (p2) by electroporation at 90 days of gestation.
  • Table 3 demonstrates the weight (kg) of control animals born from an uninjected sow (p3) at the same date.
  • Table 4 shows body composition data (fat%>/BW/d mean) of the piglets from the pSP-
  • HV-GHRH-injected sow HV-GHRH-injected sow and the uninjected sow.
  • This table represents the relative proportion of fat to body weight and shows piglets from the injected sow had 18.5% less fat per unit of weight. Pigs p2/l and p2/6 were sacrificed before the body composition data was obtained. Biochemistry of the piglets was similar to that demonstrated for the second pregnancy of this sow (see Example 15). The p values are very significant at all time points.
  • Table 5 demonstrates the weight data from the second litter of the sow injected with pSP-HV-GHRH during the first pregnancy. TABLE 5: PIGLET BODY COMPOSITION OVER TIME
  • TELAZOL a mixture of tiletamine hydrochloride and zolazepam hydrochloride
  • TELAZOL a mixture of tiletamine hydrochloride and zolazepam hydrochloride
  • ketamine/xylazine HC1 for the anesthesia was utilized during assessment of body composition, when the piglets must lay still on their backs in a Dual X-ray Densitometry (DEXA) machine for about 15 minutes.
  • DEXA Dual X-ray Densitometry
  • ketamine 20 mg/kg + xylazine 1 mg/kg (the regular xylazine dosage is 2 mg/kg) is used.
  • a different anesthetic known in the art is administered, such as ketamine 15 mg/kg + acepromazine 0.4 mg/kg.
  • no anesthesia in the piglets is necessary to take blood, inject, etc.
  • Atropine is sometimes administered.
  • Atropine is an anticholinergic medication that is utilized frequently prior to anesthesia and is thought to facilitate the drying of secretions and to reduce the amount of required anesthetic, prevent cardiac arrhythmias during the procedure, and increase animal comfort during anesthetic recovery, with a decrease in the frequency of undesirable abnormal thermal episodes.
  • atropine there is a pretreatment with atropine at 0.05 mg/kg subq (subcutaneous).
  • Other similar drugs known in the art may be used as an alternative to atropine.
  • the IGF-I assay was performed on 5-25-00 (see Table 7). The average of the experimental group is 145.509 ng/ml and the average of all previous control groups tested is 53.08 ng/ml. Therefore, the p value is very significant (p ⁇ .0001). Given that GH stimulates production and release of IGF-I, the IGF-I assay is indicative of increases in GHRH levels and is commonly used in the art as such.
  • IGF-BP3 IGF-binding protein 3
  • IRMA Immunoradiometric Assay
  • the IRMA is a non- competitive assay in which the analyte to be measured is "sandwiched" between two antibodies. The first antibody is immobilized to the inside walls of the tubes. The other antibody is radiolabelled for detection.
  • Table 9 demonstrates total protein concentration (g/dl). The average ofthe experimental group is 5.3 g/dl, whereas the average of all previous control groups tested is 4.02 g/dl. There is very high statistical significance, with p ⁇ 0.0001.
  • Table 10 demonstrates creatinine concentrations (mg/dl). The average of the experimental group is 0.936 mg/dl, whereas the average of all previous control groups tested is 0.982 mg/dl. There is no statistical significance (p ⁇ 0.34), which is indication of normal kidney function.
  • Table 11 demonstrates BUN (blood urea levels) (mg/dl). The average of the experimental group is 3.88 mg/dl, whereas the average of all previous control groups tested is 8.119 mg/dl. There is remarkable statistical significance, with p ⁇ 0.0012.
  • Table 12 shows glucose concentrations (mg/dl). The average of the experimental group is 123.23 mg/dl, whereas the average of all previous control groups tested is 122.8 mg/dl. There is no statistical significant (p ⁇ 0.67). The term G.H. stands for gross hemo lysis; in these samples the determination ofthe biochemical constant was not possible.
  • the IGF, IGF-BP3 are increased (as a result of stimulation of GH axis), the urea and total proteins are decreased and increased respectively (which is a sign of improved protein catabolism), while insulin and glucose are maintained normal.
  • the normal levels of insulin and glucose is an advantage to the present invention, because the classical GH therapies create a "diabetes" like situation, with hyperglycemia. Creatinine, which was normal in this experiment, is a parameter used to measure the renal function which can sometimes be impaired in animals under inappropriate metabolic conditions.
  • piglets bom from multiple subsequent pregnancies to the pregnancy in which the sow was first injected with pSP-HV-GHRH show an increase in growth over normal levels or animals born from sows non- injected with DNA encoding GHRH of any form.
  • a pregnancy in pigs lasts for about 114 days, and allowing for time for lactation permits no more than 2 pregnancies /year.
  • nucleic acid encoding GHRH into a female or mother is associated with an approximately 25-50% increase of GH- producing cells.
  • a nonpregnant sow is injected prior to pregnancy.
  • other growth hormone releasing hormone analogs may be utilized, which are well known in the art.
  • wild type GHRH are used. The experiments are performed similarly to the teachings provided herein.
  • the pituitaries from the piglets are collected upon sacrifice and assayed for changes in the pituitary content. That is, the piglets will be killed and the pituitaries collected when they arrive at the market weight ( ⁇ 100kg).
  • the assays include pituitary relative content of the different types of hormone secreting cells (relative proportion of cells secreting growth hormone, prolactine, follicle stimulating hormone (FSH), etc.)
  • more sows such as about 20, are injected with the same or similar treatments as provided in Examples 14 and 15.
  • Multiple plasmid quantities are tested, such as from 100 micrograms to 10 milligrams, with groups of 5 sows utilized per treatment. The decedents are compared with the offspring of uninjected sows.
  • these experiments are performed on a farm, so the data could be standardized to that in the literature.
  • a method to increase milk production comprising the step of introducing an effective amount of a vector into cells of an animal under conditions wherein a nucleotide sequence encoding a growth hormone releasing hormone is expressed and wherein said vector comprises a promoter; the nucleotide sequence encoding said growth hormone releasing hormone; and a 3' untranslated region linked operatively for functional expression of said nucleotide sequence, and wherein said introduction and expression of said vector results in an increase in milk production of the animal.
  • the animal is a human, cow, pig, goat or sheep.
  • a vector comprising a GHRH by into an animal by methods described herein increases milk production in the animal.
  • the animal is a female or mother or a pregnant female.
  • the offspring of the female or mother grow faster in about the first two weeks due to the increase in milk production in the female or mother.
  • the increase in milk production occurs upon single injection of nucleic acid encoding a GHRH into an animal.
  • Milk samples are expressed manually at the time of farrowing (colostrum) and on day 13 and day 20 of lactation.
  • An intramuscular injection of 40 IU of oxytocin is administered (except for colostrum collection) and two glands per sow are milked as rapidly as possible until no more milk is given.
  • the samples from the two glands are mixed thoroughly and aliquots deposited in two vials with a preservative agent, such as potassium dichromate. Vials are frozen until analysis.
  • Milk fat, dry matter and protein is determined according to standard procedures in the art, such as A.O.A.C. (1980) procedures.
  • milk lactose is analyzed by a semi-automated (model 27 industrial analyzer, Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio) enzymatic procedure
  • the milk yield of each sow is determined on days 13 and 20, in a specific embodiment, by weighing the pigs at hourly intervals before and after nursing as described by Lewis et al (1978) and Mahan et al.
  • milk yield is calculated by multiplying by four the yield obtained during the subsequent 6 hours.
  • ligands for the growth hormone secretagogue receptor give a similar result as delivery of a GHRH nucleic acid.
  • GHS-R growth hormone secretagogue receptor
  • MK-0677 from Merck (Whitehouse Station, NJ)
  • GHRP-6 for review see Bowers, 1998)
  • ghrelin an endogenous ligand
  • hexarelin Europeptides
  • L-692,943 Merck & Co.; Whitehouse Station, NJ
  • NN703 Novo Nordisk; Bagsvaerd, Denmark
  • any compound which acts as an agonist on the GHS-R receptor all of which are well known to a skilled artisan (see, for example, Pong et al. (1996); Howard et al. (1996); or Smith et al. (1997)).
  • GHS-R is upstream of GHRH and increases GHRH release from the pituitary gland.
  • a GHS-R ligand is given orally (such as by adding to the feed or drinking water), which would amplify the effects of GHRH on causing release of GH from the pituitary gland.
  • the GHRH nucleic acid delivery of the present invention would get an added enhancement.
  • the inventors propose that a likely mechanism of action is that the additional GHRH produces increases in the expression of pit- 1 (a transcription factor involved in development of GH producing cells, somatotrophs, in anterior pituitary during embryogenesis).
  • the GHRH nucleic acid delivery of the present invention is administered in combination with at least one GHS-R ligand.
  • the GHS-R ligand is administered in a pharmaceutically acceptable composition
  • the ectopically-produced GHRH in a pregnant animal passes through the placenta to the offspring and enhances long term GH production in progeny, which then exhibit increased growth and changed body composition.
  • the injected sows produce significantly more milk.
  • plasmid injection was followed by electroporation using a 6-needle array electrode and conditions as described in herein and (Draghia-Akli et al, 1999). Six matched sows were used as controls. The animals gave birth within 24 hours of each other. A total of 132 piglets were analyzed in the subsequent studies.
  • Piglets were weaned at 21 days and analyzed to slaughter weight, at 170 days after birth. Piglets from injected sows were on average 18%> bigger at weaning (FIG. 9). Half of each litter was cross-fostered to either control sows (piglets from injected sows) or injected sows (piglets from control sows). Interestingly, controls cross-fostered to injected animals were significantly bigger (to up to 12.2%) than their littermates, p ⁇ 0.02 (FIG. 10). This change in weight in control animals cross-fostered to GHRH treated animals is indicative of the significantly increased milk production in the injected sows.
  • Creatinine concentration was normal (0.936 mg/dl versus controls 0.982 mg/dl, p ⁇ .34), which is indication of a normal kidney function.
  • the insulin levels were normal.
  • the normal level of insulin and glucose is an advantage because the classical GH therapies create a "diabetes"-like situation, with hyperglycemia (Pursel et al, 1990).
  • Regulated tissue/fibre-type-specific hGH- containing plasmids were previously used for the delivery and stable production of GH in livestock and GH-deficient hosts by either transgenesis, myoblast transfer or liposome- mediated intravenous injection (Dahler et al, 1994; Pursel et al, 1990; Barr and Leiden, 1991).
  • the plasmid pSPc5-12 contains a 360bp SacI/BamHI fragment of the SPc5-12 synthetic promoter in the SacI/BamHI sites of pSK-GHRH backbone (Draghia-Akli et al, 1997).
  • the wild type porcine GHRH was obtained by sire directed mutagenesis of human GHRH cDNA (1-40)OH at positions 34: Ser to Arg, 38: Arg to Glu; the mutated porcine HV-GHRH DNA was obtained by site directed mutagenesis of human GHRH cDNA (1-40)OH at positions 1 : Tyr to His, 2 Ala to Val, 15: Gly to Ala, 27: Met to Leu, 28: Ser to Asn, 34: Ser to Arg, 38: Arg to Glu (Altered Sites II in vitro Mutagenesis System, Promega, Madison, Wl), and cloned into the BamHI/ Hind III sites of pSP-GHRH.
  • the GHRH cDNA was followed by the 3 ' untranslated region of human growth hormone, to create pSPc5- 12- wt-GHRH and pSPc5-12-HV-GHRH.
  • the control plasmid contained the E. coli beta-galactosidase gene under the control of the same synthetic promoter to create pSP-bgal.
  • a total of 10 mg plasmid was injected directly into the left semitendinosous muscle of pigs. Two minutes later, the injected muscle was electroporated using 6-needle array injectable electrodes, 1 cm diameter, 22 gauge, 2 cm length, using the following conditions: 6 pulses, alternate field in between needles, 200V/cm, 60 milliseconds/ pulse, as described (Draghia-Akli et al, 1999; Aihara and Miyazai, 1998).
  • Porcine IGF-I RIA Porcine IGF-I was measured by heterologous human IGF-I assay (Diagnostic System Lab., Webster, TX).
  • Porcine Insulin RIA Porcine insulin was measured by heterologous human assay (Linco Research Inc.; St. Charles, Missouri). The sensitivity of the assay was 2 microU/ml.
  • Body composition data Weights were measured on the same calibrated scales (certified to have an accuracy to ⁇ .2kg and a coefficient of variation of 0.3%>) throughout the study, twice a week.
  • GH growth hormone
  • GHRH growth hormone releasing hormone
  • SS stomatostatin
  • GH pulses are a result of GHRH secretion that are associated with a diminution or withdrawal of somatostatin secretion.
  • the pulse generator mechanism appears to be timed by GH-negative feedback.
  • ghrelin a novel peptide initially isolated from the rat stomach, has been recognized as an important regulator of GH secretion and energy homeostasis.
  • Ghrelin is the endogenous ligand of the growth hormone secretagogue receptor and its GH-releasing activity in vivo is dependent on GHRH (Hataya et al, 2001).
  • GH is released in a highly regulated, distinctive pulsatile pattern, which occurs 4-8 times within 24 h, and has profound importance for its biological activity (Argente et al, 1996).
  • the episodic pattern of secretion relates to the optimal induction of physiological effects at a peripheral level (Veldurs, 1998).
  • the expression, processing, and/or release of GH isoforms and the relative proportion in between them are under differential control during growth and developmental stage (Araburo et al, 2000).
  • somatotrophs also depend upon paracrine processes within the pituitary itself and involve growth factors and several neuropeptides, for instance, vasoactive intestinal peptide (Rawlings et al, 1995), angiotensin 2, endothelin (Tomic et al, 1999), and activin (Billesbup et al, 1990).
  • Effective and regulated expression of the GH and insulin-like growth factor I (IGF-I) pathway is essential for optimal linear growth, homeostasis of carbohydrate, protein, and fat metabolism, and for providing a positive nitrogen balance (Murray and Shalet, 2000).
  • GHRH, GH, ghrelin, prolactin (PRL) and IGF-I play a significant role in regulation of the humoral and cellular immune responses in physiological as well as pathological situations (Geffher et al, 1997; Hattori et al, 2001).
  • GHRH Hypothalamic tissue-specific expression of the GHRH gene is not required for activity, as extra-cranially secreted GHRH can be biologically active (Faglia et al, 1992; Melmed, 1991).
  • Pathological GHRH stimulation (irrespective of its source, from transgenic models to pancreatic tumors) of GH activity can result in proliferation, hyperplasia, and adenoma of adenohypophysial cells (Asa et al, 1992; Sano et al, 1988). Nevertheless, the long-term effects of a sustained GHRH treatment on the offspring of the animals receiving the therapy is yet unknown.
  • the ectopically-produced GHRH in a pregnant animal passes through the placenta to the offspring, determines pituitary hyperplasia and enhances long term GH production in progeny, which would then exhibit increased growth and changed body composition.
  • the offspring of a GHRH myogenic vector injection into a mammal pregnant rats were injected with 30 ⁇ g of plasmid DNA pSP-HV-GHRH or pSP- ⁇ gal at 16 days of gestation. The injection was followed by electroporation, to enhance plasmid uptake.
  • Electrogene therapeutic transfer allows genes to be efficiently transferred and expressed in desired organs or tissues, and it is capable of providing long-term expression following a single administration. This method may represent a new approach for highly effective nucleic acid transfer that does not require viral genes or particles.
  • GHRH the upstream stimulator of GH
  • the use of GHRH is an alternate strategy that may increase not only growth performance or milk production, but more importantly, the efficiency of production from both practical and metabolic perspectives (Dubreuil et al, 1990).
  • the high cost of the recombinant peptides and the required frequency of administration currently limit the widespread use of this treatment.
  • These major drawbacks can be obviated by using a nucleic acid transfer approach to direct the ectopic production of GHRH, particularly when its production is sustained chronically.
  • the plasmid pSPc5-12 contains a 360bp SacI/BamHI fragment ofthe SPc5-12 synthetic promoter (Li et al, 1999) in the SacI/BamHI sites of pSK- GHRH backbone (Draghia-Akli et al, 1999).
  • the mutated porcine GHRH cDNA were obtained by site-directed mutagenesis of human GHRH cDNA (Altered Sites II in vitro Mutagenesis System, Promega, Madison, Wl).
  • the mutated 228-bp fragment of porcine GHRH (part of exon 2, all exon 3 and part of exon 4), which encodes the 31 amino acid signal peptide and a mutated porcine GHRH (1-40)OH, is characterized by the following amino acid substitutions: Gly 15 to Ala, Met27 to Leu and Ser28 to Asn, and conversion of Tyrl to His, and Ala2 to Val.
  • This fragment was cloned into the BamHI/ Hind III sites of pSK-GHRH.
  • hGH pA is a 3 ' untranslated region and poly(A) signal from the human GH gene. Plasmids were grown in E. coli DH5 ⁇ (Gibco BRL, Carlsbad, CA). Endotoxin-free plasmid (Qiagen Inc., Chatsworth, CA, USA) preparations were diluted in PBS, pH 7.4 to 1 mg/ml.
  • the left tibialis anterior muscle of rats was injected with 30 mg of pSP-HV-GHRH in 100 ml PBS using 0.3 cc insulin syringes (Becton-Dickinson, Franklin Lakes, NJ). Control animals were injected with PBS. For both groups, the injection was followed by caliper electroporation, as described (Draghia-Akli et al, 1999). Briefly, two minutes after injection, the rat leg was placed in between a two needles electrode, 1 cm length, 26 gauge, 1 cm in between needles (Genetronics, San Diego, CA) and electric pulses were applied to the area.
  • Offspring studies All injected rats gave birth at 20-22 days of gestation. In the first study 240 offspring and in the second study 60 offspring were analyzed from birth to 5 month of age (birth, 2, 3, 6, 8, 12, 16, 22 weeks after birth). Body weights were recorded at these time points using the same calibrated balance. At the end of the experiment, body composition was performed post-mortem. Blood was collected, centrifuged immediately at 0°C, and stored at -80°C prior to analysis.
  • Organs (heart, liver, spleen, kidney, pituitary, brain, adrenals, skeletal muscles - tibialis anterior (TA), gastrocnemius (G), soleus (S), and extensor digitorum longus (EDL), carcass, fat from injected animals and controls were removed, weighed on an analytical balance and snap frozen in liquid nitrogen. Tibia length was measured and recorded.
  • Rat IGF-I Radioimmunoassay Rat IGF-I was measured by specific radioimmunoassay (Diagnostic System Laboratories, Webster, Texas). The sensitivity of the assay was 0.8 ng/ml; intra-assay and inter-assay variation was 2.4%> and 4.1%> respectively.
  • GRF Growth hormone-releasing peptide
  • Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402(6762):656-60.

Abstract

La croissance est améliorée au moyen d'une technique d'amélioration du potentiel de la croissance, laquelle technique consiste à administrer une séquence d'acides aminés, telle que GHRH ou un analogue, à un animal femelle, de préférence par administration par voie parentérale. Les porcelets nés de truies auxquelles on a injecté un ADN codant pour GHRH, sont plus gros. Ces effets sont démontrés lors de gestations ultérieures sans administration(s) supplémentaire(s) du vecteur.
PCT/US2001/048726 2000-12-12 2001-12-12 Administration d'une sequence d'acides amines a un animal femelle WO2002061037A2 (fr)

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KR10-2003-7007872A KR20040039187A (ko) 2000-12-12 2001-12-12 암컷 동물에 핵산 서열의 투여
CA2430921A CA2430921C (fr) 2000-12-12 2001-12-12 Utilisation d'un vecteur codant pour un peptide de ghrh destine a ameliorer la croissance dans de multiples portees de la progeniture d'animaux femelles
MXPA03005236A MXPA03005236A (es) 2000-12-12 2001-12-12 Administracion de secuencia de acido nucleico a un animal hembra para mejorar el crecimiento en la progenie.
EP01997073A EP1364004A4 (fr) 2000-12-12 2001-12-12 Administration d'une sequence d'acides amines a un animal femelle
AU2002248194A AU2002248194B2 (en) 2000-12-12 2001-12-12 Administration of nucleic acid sequence to female animal
BR0116472-4A BR0116472A (pt) 2000-12-12 2001-12-12 Administração de sequência de ácido nucléico a animal fêmea para aumentar o crescimento de prole

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004022102A2 (fr) * 2002-09-02 2004-03-18 Avontec Gmbh Formulation pour introduire des acides nucleiques dans des eucaryotes
WO2004067719A2 (fr) * 2003-01-28 2004-08-12 Advisys, Inc. Reduction de reforme dans l'hormone de liberation de l'hormone de croissance (ghrh) d'animaux de troupeau
EP1450605A2 (fr) * 2001-10-26 2004-09-01 Baylor College Of Medicine Composition et methode permettant de modifier la masse maigre et les proprietes osseuses d'un sujet
EP1499731A2 (fr) * 2002-02-07 2005-01-26 Baylor College Of Medicine Developpement modifie de l'hypophyse chez la progeniture de femelles animales portantes traitees par la therapie hormonale de liberation de l'hormone de croissance
EP1543020A2 (fr) * 2002-07-16 2005-06-22 Advisys, Inc. Plasmides synthetiques optimises par codons
EP1573004A2 (fr) * 2002-11-04 2005-09-14 Advisys, Inc. Promoteurs musculaires de synthese dotes d'activites depassant celles des sequences regulatrices d'origine naturelle dans des cellules cardiaques
CN104031930A (zh) * 2014-05-30 2014-09-10 华南农业大学 一种提高母猪乳中营养及免疫物质含量的方法
EP3787415A4 (fr) * 2018-04-29 2021-12-29 Kalmarna Limited Compositions orale et méthodes pour modifier la descendance de mammifères

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102127545A (zh) * 2010-11-24 2011-07-20 山东农业大学 一种骨骼肌特异性ckm启动子及其应用
CN105219774B (zh) * 2015-10-10 2019-04-30 广西大学 猪胰岛素特异性表达启动子pip2及其应用
CN105617404A (zh) * 2016-01-27 2016-06-01 广州市科虎生物技术研究开发中心 Grf表达质粒在制备降低仔猪出生弱仔率的药物中的用途

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6165755A (en) * 1997-01-23 2000-12-26 University Of Victoria Innovation And Development Corporation Chicken neuropeptide gene useful for improved poultry production
US6423693B1 (en) * 1997-07-24 2002-07-23 Baylor College Of Medicine Growth hormone releasing hormone expression system and methods of use, including use in animals

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2622455B1 (fr) * 1987-11-04 1991-07-12 Agronomique Inst Nat Rech Application du facteur de stimulation de la secretion de l'hormone de croissance humaine, de ses fragments actifs et des analogues correspondants, pour augmenter la production laitiere et le poids des nouveau-nes chez les mammiferes
ZA914983B (en) * 1990-06-29 1992-03-25 Hoffmann La Roche His-grf-analogs
EP1204321B1 (fr) * 1999-07-26 2005-11-09 Baylor College Of Medicine Analogue superactif de la somatostatine porcine

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6165755A (en) * 1997-01-23 2000-12-26 University Of Victoria Innovation And Development Corporation Chicken neuropeptide gene useful for improved poultry production
US6423693B1 (en) * 1997-07-24 2002-07-23 Baylor College Of Medicine Growth hormone releasing hormone expression system and methods of use, including use in animals

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GONZALEZ L. ET AL.: 'Up-regulation of GH-binding protein by mouse GH in transgenic mice overexpressing GH-releasing hormone' JOURNAL OF ENDOCRINOLOGY vol. 163, no. 2, November 1999, pages 299 - 307, XP002966757 *
KOVACS M. ET AL.: 'Effects of antagonists of growth hormone-releasing hormone (GHRH) on GH and insulin-like growth factor I levels in transgenic mice-overexpressing the human GHRH gene, an animal model of acromegaly' ENDOCRINOLOGY vol. 138, no. 11, November 1997, pages 4536 - 4542, XP002966759 *
See also references of EP1364004A2 *
WOJTKIEWICZ P.W. ET AL.: 'Transcript abundance in mouse pituitaries with altered growth hormone expression quantified by reverse transcriptase polymerase chain reaction implicates transcription factor Zn-16 gen regulation in vivo' ENDOCRINOLOGY vol. 18, no. 1, June 2002, pages 67 - 74, XP002966758 *

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EP1450605A2 (fr) * 2001-10-26 2004-09-01 Baylor College Of Medicine Composition et methode permettant de modifier la masse maigre et les proprietes osseuses d'un sujet
US7338656B2 (en) 2001-10-26 2008-03-04 Baylor College Of Medicine Composition and method to alter lean body mass and bone properties in a subject
EP1450605A4 (fr) * 2001-10-26 2007-01-17 Baylor College Medicine Composition et methode permettant de modifier la masse maigre et les proprietes osseuses d'un sujet
EP1499731A4 (fr) * 2002-02-07 2006-04-26 Baylor College Medicine Developpement modifie de l'hypophyse chez la progeniture de femelles animales portantes traitees par la therapie hormonale de liberation de l'hormone de croissance
EP1499731A2 (fr) * 2002-02-07 2005-01-26 Baylor College Of Medicine Developpement modifie de l'hypophyse chez la progeniture de femelles animales portantes traitees par la therapie hormonale de liberation de l'hormone de croissance
US7250405B2 (en) 2002-02-07 2007-07-31 Baylor College Of Medicine Modified pituitary gland development in offspring from expectant mother animals treated with growth hormone releasing hormone therapy
EP1543020A4 (fr) * 2002-07-16 2006-11-22 Advisys Inc Plasmides synthetiques optimises par codons
US7316925B2 (en) 2002-07-16 2008-01-08 Vgx Pharmaceuticals, Inc. Codon optimized synthetic plasmids
EP1543020A2 (fr) * 2002-07-16 2005-06-22 Advisys, Inc. Plasmides synthetiques optimises par codons
CN100346833C (zh) * 2002-09-02 2007-11-07 亚文塔克有限公司 转移核酸到真核生物细胞内所用的含非甾体抗炎药和核酸的药物配制品
WO2004022102A2 (fr) * 2002-09-02 2004-03-18 Avontec Gmbh Formulation pour introduire des acides nucleiques dans des eucaryotes
WO2004022102A3 (fr) * 2002-09-02 2004-05-13 Avontec Gmbh Formulation pour introduire des acides nucleiques dans des eucaryotes
EP1573004A4 (fr) * 2002-11-04 2006-08-09 Advisys Inc Promoteurs musculaires de synthese dotes d'activites depassant celles des sequences regulatrices d'origine naturelle dans des cellules cardiaques
EP1573004A2 (fr) * 2002-11-04 2005-09-14 Advisys, Inc. Promoteurs musculaires de synthese dotes d'activites depassant celles des sequences regulatrices d'origine naturelle dans des cellules cardiaques
AU2003295366B2 (en) * 2002-11-04 2011-11-24 Advisys, Inc. Synthetic muscle promoters with activities exceeding naturally occurring regulatory sequences in cardiac cells
WO2004067719A2 (fr) * 2003-01-28 2004-08-12 Advisys, Inc. Reduction de reforme dans l'hormone de liberation de l'hormone de croissance (ghrh) d'animaux de troupeau
WO2004067719A3 (fr) * 2003-01-28 2005-03-31 Advisys Inc Reduction de reforme dans l'hormone de liberation de l'hormone de croissance (ghrh) d'animaux de troupeau
CN104031930A (zh) * 2014-05-30 2014-09-10 华南农业大学 一种提高母猪乳中营养及免疫物质含量的方法
EP3787415A4 (fr) * 2018-04-29 2021-12-29 Kalmarna Limited Compositions orale et méthodes pour modifier la descendance de mammifères

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BR0116472A (pt) 2005-04-05
WO2002061037B1 (fr) 2004-01-15
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CN1575301A (zh) 2005-02-02
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