Canine Specific Growth Hormone Releasing Hormone

Abstract

A composition and a method of increasing growth hormone ("GH") values in a canine or dog, and more specifically, a canine- or dog-specific growth hormone releasing hormone ("dGHRH"), or functional biological equivalent thereof. The dGHRH is an isolated composition or a nucleic acid molecule that encodes the dGHRH or functional biological equivalent. Also, a method for delivering the composition of this invention to a subject, wherein the dGHRH increases the level of growth hormone ("GH") secretion in a recipient subject, such as a canine or dog.

Description

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Ser. No. 60/492,427, entitled "Canine Specific Growth Hormone Releasing Hormone," filed on Aug. 4, 2003, having Draghia-Akli, listed as the inventor, the entire content of which is hereby incorporated by reference.

BACKGROUND

[0002] This invention pertains to an isolated composition and a method of increasing growth hormone ("GH") values in a canine or dog. More specifically, the invention pertains to a canine- or dog-specific growth hormone releasing hormone ("dGHRH"), or functional biological equivalent thereof. The dGHRH is an isolated composition or a nucleic acid molecule that encodes the dGHRH or functional biological equivalent. Another aspect of the current invention includes a method for delivering the composition of this invention to a subject, wherein the dGHRH increases the level of growth hormone ("GH") secretion in a recipient subject, such as a canine or dog.

[0003] In the United States, the companion canine population is about 60 million. Although not wanting to be bound by theory, the average lifespan for these canines has increased in recent decades due to better nutrition, and better health care options. Even though the average disease profile and lifespan of the canine population are generally breed specific, there are common disease related features and age related features that are present in most mammals. For example, as mammals age, the GHRH-GH-IGF-I axis undergoes considerable decrement, with reduced GH secretion and IGF-I production associated with a loss of skeletal muscle mass (sarcopenia), osteoporosis, increased fat deposition, decreased lean body mass, and other disorders. Studies in humans and other mammals have demonstrated that the development of these changes can be offset by recombinant growth hormone ("GH") therapy. One benefit of the claimed invention is observed when a dog specific growth hormone releasing hormone ("dGHRH") composition is delivered to a canine subject and the level of GH secretion in the canine subject is increased. Another aspect of the current invention is the dGHRH molecule or functional biological equivalent thereof. The composition may also be a nucleic acid molecule that encodes the dGHRH or functional biological equivalent thereof. The dGHRH can be defined as a biologically active polypeptide that has been engineered to contain a distinct amino acid sequence having similar or improved biologically activity when compared to a generic GHRH ("GHRH") polypeptide. Other benefits from administering the dGHRH compound to the canine subject are outlined in preferred embodiments and include: increased insulin-like growth factor I ("IGF-I"), increased red blood cells production and hemoglobin concentration, and improved protein metabolism.

[0004] In humans and other mammals, regulated expression of the GH pathway is considered essential for optimal linear growth, as well as homeostasis of carbohydrate, protein, and fat metabolism. GH synthesis and its pulsatile secretion from the anterior pituitary is stimulated by GHRH and inhibited by somatostatin, both hypothalamic hormones (Frohman et al., 1992). GH increases production of insulin-like growth factor-I ("IGF-I") primarily in the liver, as well as other target organs. IGF-I and GH feedback on the hypothalamus and pituitary to inhibit GHRH release and GH secretion. The endogenous rhythm of GH secretion becomes entrained to the imposed rhythm of exogenous GHRH (Caroni and Schneider, 1994).

[0005] Although not wanting to be bound by theory, the linear growth velocity and body composition of humans, farm animals, and companion animals appear to respond to GH or GHRH replacement therapies under a broad spectrum of conditions. Similarly, anemia associated with different diseases and conditions can be treated by physiologically stimulating the GHRH axis (Draghia-Akli et al., 2002a; Draghia-Akli et al., 2003a). However, the etiology of these conditions can vary significantly. For example, in 50% of human GH deficiencies the GHRH-GH-IGF-I axis is functionally intact, but does not elicit the appropriate biological responses in its target tissues. Similar phenotypes are produced by genetic defects at different points along the GH axis (Parks et al., 1995), as well as in non-GH-deficient short stature. In humans, these non-GH-deficiency causes of short stature, such as Turner syndrome (Butler et al., 1994), hypochondroplasia (Foncea et al., 1997), Crohn's disease (Parrizas and LeRoith, 1997), intrauterine growth retardation (Hoess and Abremski, 1985) or chronic renal insufficiency (Lowe, Jr. et al., 1989) can be efficiently treated with GHRH or GH therapy (Gesundheit and Alexander, 1995). In companion animals, such as dogs, there is little or no available therapy, and recombinant protein therapies have proved to be inefficient (Kooistra et al., 1998; Kooistra et al., 2000; Rijnberk et al., 1993).

[0006] In aging mammals, the GHRH-GH-IGF-I axis undergoes considerable decrement having reduced GH secretion and IGF-I production associated with a loss of skeletal muscle mass (sarcopenia), osteoporosis, increased fat deposition and decreased lean body mass (Caroni and Schneider, 1994; Veldhuis et al., 1997). It has been demonstrated that the development of these changes can be offset by recombinant GH therapy. GH replacement therapy both in children and the elderly is widely used clinically. Current GH therapy has several shortcomings, however, including frequent subcutaneous or intravenous injections, insulin resistance and impaired glucose tolerance (Rabinovsky et al., 1992); children are also vulnerable to premature epiphyseal closure and slippage of the capital femoral epiphysis (Liu and LeRoith, 1999). A "slow-release" form of GH (from Genentech) has been developed that only requires injections every 14 days. However, this GH product appears to perturb the normal physiological pulsatile GH profile, and is also associated with frequent side effects.

[0007] Various GH and GHRH regimens are also available for use in domestic livestock. For example, administration of GHRH and GH stimulate milk production, with an increase in feed to milk conversion. This therapy enhances growth primarily by increasing lean body mass (Lapierre et al., 1991; van Rooij et al., 2000) with overall improvement in feed efficiency. Hot and chilled carcass weights are increased and carcass lipid (percent of soft-tissue mass) is decrease by administration of GHRH and GH (Etherton et al., 1986).

[0008] Administering novel GHRH analog proteins (U.S. Pat. Nos. 5,847,066; 5,846,936; 5,792,747; 5,776,901; 5,696,089; 5,486,505; 5,137,872; 5,084,442, 5,036,045; 5,023,322; 4,839,344; 4,410,512, RE33,699), synthetic or naturally occurring peptide fragments of GHRH (U.S. Pat. Nos. 4,833,166; 4,228,158; 4,228,156; 4,226,857; 4,224,316; 4,223,021; 4,223,020; 4,223,019) for the purpose of increasing release of GH have been reported. A GHRH analog containing the following mutations has been reported (U.S. Pat. No. 5,846,936): Tyr at position 1 to His; Ala at position 2 to Val, Leu, or others; Asn at position 8 to Gln, 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 is the subject of U.S. Pat. No. 6,551,996 ("the '996 patent"), issued on Apr. 22, 2003 having Schwartz et al., listed as inventors. The '996 patent teaches application of a GHRH analog containing mutations that improve the ability to elicit the release of GH. In addition, the '996 patent relates to the treatment of growth deficiencies; the improvement of growth performance; the stimulation of production of GH in an animal at a greater level than that associated with normal growth; and the enhancement of growth utilizing the administration of GH releasing hormone analog and is herein incorporated by reference.

[0009] Studies in humans, sheep or pigs showed that continuous infusion with recombinant GHRH protein restores the normal GH pattern without desensitizing GHRH receptors or depleting GH supplies as this system is capable of feed-back regulation, which is abolished in the GH therapies (Dubreuil et al., 1990; Vance, 1990; Vance et al., 1985). Although GHRH protein therapy stimulates normal cyclical GH secretion with virtually no side effects (Corpas et al., 1993), the short half-life of the molecule in vivo requires frequent (e.g. one to three times per day) intravenous, subcutaneous or intranasal administrations at about a 300-fold higher dose. Thus, recombinant GHRH administration is not practical as a chronic therapy. However, extracranially secreted GHRH, as a mature or a truncated polypeptide, is often biologically active (Thorner et al., 1984) and a low level of serum GHRH (100 pg/ml) stimulates GH secretion (Corpas et al., 1993). These characteristics make GHRH an excellent candidate for plasmid mediated supplementation of a gene product.

[0010] Transgene Delivery and in vivo Expression: Although not wanting to be bound by theory, the delivery of specific transgenes to somatic tissue to correct inborn or acquired deficiencies and imbalances is possible. Such transgene-based drug delivery offers a number of advantages over the administration of recombinant proteins. These advantages include: the conservation of native protein structure; improved biological activity; avoidance of systemic toxicities; and avoidance of infectious and toxic impurities. Because the protein is synthesized and secreted continuously into the circulation, plasmid mediated therapy allows for prolonged production of the protein in a therapeutic range. In contrast, the primary limitation of using recombinant protein is the limited availability of protein after each administration.

[0011] In a plasmid-based expression system, a non-viral transgene vector may comprise of a synthetic transgene delivery system in addition to the nucleic acid encoding the therapeutic genetic product. In this way, the risks associated with the use of most viral vectors can be avoided, including the expression of viral proteins that can induce immune responses against target tissues and the possibility of DNA mutations or activations of oncogenes. The non-viral expression vector products generally have low toxicity due to the use of "species-specific" components for gene delivery, which minimizes the risks of immunogenicity generally associated with viral vectors. Additionally, no integration of plasmid sequences into host chromosomes has been reported in vivo to date, so that this type of nucleic acid vector therapy should neither activate oncogenes nor inactivate tumor suppressor genes. As episomal systems residing outside the chromosomes, plasmids have defined pharmacokinetics and elimination profiles, leading to a finite duration of gene expression in target tissues.

[0012] Direct plasmid DNA gene transfer is currently the basis of many emerging nucleic acid therapy strategies and does not require viral components or lipid particles (Aihara and Miyazaki, 1998; Muramatsu et al., 2001). Skeletal muscle is target tissue, because muscle fiber has a long life span and can be transduced by circular DNA plasmids that are expressed in immunocompetent hosts (Davis et al., 1993; Tripathy et al., 1996). Plasmid DNA constructs are attractive candidates for direct therapy into the subjects skeletal muscle because the constructs are well-defined entities that are biochemically stable and have been used successfully for many years (Acsadi et al., 1991; Wolff et al., 1990). The relatively low expression levels of an encoded product that are achieved after direct plasmid DNA injection are sometimes sufficient to indicate bio-activity of secreted peptides (Danko and Wolff, 1994; Tsurumi et al., 1996). Previous reports demonstrated that human GHRH cDNA could be delivered to muscle by an injectable myogenic expression vector in mice where it transiently stimulated GH secretion to a modest extent over a period of two weeks (Draghia-Akli et al., 1997).

[0013] Efforts have been made to enhance the delivery of plasmid DNA to cells by physical means including electroporation, sonoporation, and pressure. Although not wanting to be bound by theory, the administration of a nucleic acid construct by electroporation involves the application of a pulsed electric field to create transient pores in the cellular membrane without causing permanent damage to the cell, which allows exogenous molecules to enter the cell (Smith and Nordstrom, 2000). By adjusting the electrical pulse generated by an electroporetic system, nucleic acid molecules can travel through passageways or pores in the cell that are created during the procedure. U.S. Pat. No. 5,704,908 describes an electroporation apparatus for delivering molecules to cells at a selected location within a cavity in the body of a patient. Similar pulse voltage injection devices are also described in U.S. Pat. Nos. 5,439,440 and 5,702,304, and PCT WO 96/12520, 96/12006, 95/19805, and 97/07826, which are hereby incorporated by reference.

[0014] Recently, significant progress to enhance plasmid delivery in vivo and subsequently to achieve physiological levels of a secreted protein was obtained using the electroporation technique. Electroporation has been used very successfully to transfect tumor cells after injection of plasmid (Lucas et al., 2002; Matsubara et al., 2001)) or to deliver the anti-tumor drug bleomycin to cutaneous and subcutaneous tumors in humans (Gehl et al., 1998; Heller et al., 1996). Electroporation also has been extensively used in mice (Lesbordes et al., 2002; Lucas et al., 2001; Vilquin et al., 2001), rats (Terada et al., 2001; Yasui et al., 2001), and dogs (Fewell et al., 2001) to deliver therapeutic genes that encode for a variety of hormones, cytokines or enzymes. Previous studies using GHRH showed that plasmid therapy with electroporation is scalable and represents a promising approach to induce production and regulated secretion of proteins in large animals and humans (Draghia-Akli et al., 1999; Draghia-Akli et al., 2002c). Electroporation also has been extensively used in rodents and other small animals (Bettan et al, 2000; Yin and Tang, 2001). It has been observed that the electrode configuration affects the electric field distribution, and subsequent results (Gehl et al., 1999; Miklavcic et al., 1998). Although not wanting to be bound by theory, needle electrodes give consistently better results than external caliper electrodes in a large animal model.

[0015] The ability of electroporation to enhance plasmid uptake into the skeletal muscle has been well documented. Similarly, plasmids formulated with poly-L-glutamate ("PLG") or polyvinylpyrrolidone ("PVP") were observed to have an increase in plasmid transfection, which consequently increased the expression of a desired transgene. For example, plasmids formulated with PLG or PVP were observed to increase gene expression to up to 10 fold in the skeletal muscle of mice, rats, and dogs (Fewell et al., 2001; Mumper et al., 1998). Although not wanting to be bound by theory, the anionic polymer sodium PLG enhances plasmid uptake at low plasmid concentrations and reduces any possible tissue damage caused by the procedure. PLG is a stable compound and it is resistant to relatively high temperatures (Dolnik et al., 1993). PLG has been used to increase stability of anti-cancer drugs (Li et al., 2000) and as "glue" to close wounds or to prevent bleeding from tissues during wound and tissue repair (Otani et al., 1996; Otani et al., 1998). PLG has been used to increase stability in vaccine preparations (Matsuo et al., 1994) without increasing their immunogenicity. PLG also has been used as an anti-toxin after antigen inhalation or exposure to ozone (Fryer and Jacoby, 1993).

[0016] Although not wanting to be bound by theory, PLG increases the transfection of the plasmid during the electroporation process, not only by stabilizing the plasmid DNA and facilitating the intracellular transport through the membrane pores, but also through an active mechanism. For example, positively charged surface proteins on the cells could complex the negatively charged PLG linked to plasmid DNA through protein-protein interactions. When an electric field is applied, the surface proteins reverse direction and actively internalize the DNA molecules, a process that substantially increases the transfection efficiency. Furthermore, PLG will prevent the muscle damage associated with in vivo plasmid delivery (Draghia-Akli et al., 2002b) and will increase plasmid stability in vitro prior to injection. There are studies directed to electroporation of eukaryotic cells with linear DNA (McNally et al., 1988; Neumann et al., 1982) (Toneguzzo et al., 1988) (Aratani et al., 1992; Nairn et al., 1993; Xie and Tsong, 1993; Yorifuji and Mikawa, 1990), but these examples illustrate transfection into cell suspensions, cell cultures, and the like, and such transfected cells are not present in a somatic tissue.

[0017] U.S. Pat. No. 4,956,288 is directed to methods for preparing recombinant host cells containing high copy number of a foreign DNA by electroporating a population of cells in the presence of the foreign DNA, culturing the cells, and killing the cells having a low copy number of the foreign DNA.

[0018] Although not wanting to be bound by theory, a GHRH cDNA can be delivered to muscle of mice and humans by an injectable myogenic expression vector where it can transiently stimulate GH secretion over a period of two weeks (Draghia-Akli et al., 1997). This injectable vector system was optimized by incorporating a powerful synthetic muscle promoter (Li et al., 1999) coupled with a novel protease-resistant GHRH molecule with a substantially longer half-life and greater GH secretory activity (pSP-HV-GHRH) (Draghia-Akli et al., 1999). Highly efficient electroporation technology was optimized to deliver the nucleic acid construct to the skeletal muscle of an animal (Draghia-Akli et al., 2002b). Using this combination of vector design and electric pulses plasmid delivery method, the inventors were able to show increased growth and favorably modified body composition in pigs (Draghia-Akli et al., 1999; Draghia-Akli et al., 2003b) and rodents (Draghia-Akli et al., 2002c). The modified GHRH nucleic acid constructs increased red blood cell production in companion animals with cancer and cancer treatment-associated anemia (Draghia-Akli et al., 2002a). In pigs, available data suggested that the modified porcine HV-GHRH was more potent in promoting growth and positive body composition changes than the wild-type porcine GHRH (Draghia-Akli et al., 1999). One aspect of the current invention describes a species-specific dGHRH expression vector that comprises a more efficient composition to increase red blood cell production in a canine subject than the protease resistant HV-GHRH molecule.

SUMMARY

[0019] Although the average disease profile and lifespan of the canine population are generally breed specific, there are common disease related features and age related features that are present in most mammals. Studies in mammals have demonstrated that the development of hematological changes can be offset by recombinant growth hormone ("GH") therapy. The current invention comprises compositions and methods for increasing GH values in canines.

[0020] One aspect of the current invention comprises a canine or dog specific growth hormone releasing hormone ("dGHRH") or functional biological equivalent thereof. In one specific embodiment, the dGHRH or functional biological equivalent increases growth hormone ("GH") when delivered into a subject. The delivered dGHRH or functional biological equivalent thereof improves hematological parameters in the subject, wherein the hematological parameters comprise: red blood cell count, hemoglobin concentration, and mean corpuscular hemoglobin.

[0021] Another aspect of the current invention comprises a nucleic acid expression construct encoding the dGHRH or functional biological equivalent thereof. In a second specific embodiment, the dGHRH or functional biological equivalent increases GH when expressed in the subject. The expressed dGHRH or functional biological equivalent thereof improves hematological parameters in the subject, wherein the hematological parameters comprise: red blood cell count, hemoglobin concentration, and mean corpuscular hemoglobin. In a third specific embodiment, the nucleic acid expression construct further comprises: a synthetic or eukaryotic promoter; a poly-adenylation signal; a selectable marker gene promoter; a ribosomal binding site; a selectable marker gene sequence; and an origin of replication. In such an arrangement, the synthetic or eukaryotic promoter, the nucleic acid sequence encoding the dGHRH or functional biological equivalent thereof, and the poly adenylation signal comprise therapeutic elements of the nucleic acid expression construct. The therapeutic elements are operatively linked and located in a first operatively-linked arrangement. Similarly, the selectable marker gene promoter, the ribosomal binding site, the selectable marker gene sequence, and the origin of replication comprise the replication elements of the nucleic acid expression vector and are operatively linked and located in a second operatively-linked arrangement. The first-operatively-linked arrangement and the second-operatively-linked arrangement comprise a circular structure of the nucleic acid expression construct, which is utilized for plasmid mediated gene supplementation. Examples of dGHRH nucleic acid expression constructs of this invention include plasmids pAV0221 and pAV00215.

[0022] Still another aspect of the current invention is a method of increasing GH values in a subject. The method comprises delivering into the subject a recombinant dGHRH or functional biological equivalent thereof. The recombinant dGHRH comprises a biologically active polypeptide, and the recombinant functional biological equivalent of dGHRH comprises a polypeptide that has been engineered to contain a distinct amino acid sequence while simultaneously having similar or improved biologically activity when compared to the dGHRH polypeptide. The increasing GH values are related by increasing hematological parameters in the subject having the delivered recombinant dGHRH or functional biological equivalent thereof.

[0023] Yet another aspect of the current invention is a method of increasing GH values in a subject. The method comprises delivering into the cells of a subject a nucleic acid expression construct that expresses the dGHRH or functional biological equivalent thereof. In a fourth specific embodiment, the nucleic acid expression construct is delivered into the cells of the subject via an electroporation method. The cells receiving the nucleic acid expression construct comprise somatic cells, stem cells, or germ cells. The dGHRH or functional biological equivalent thereof is expressed in tissue specific cells of the subject (e.g. muscle cells). Examples of nucleic acid expression constructs used for this method include plasmids pAV0221 and pAV00215. In a fifth specific embodiment, the method for delivering the dGHRH nucleic acid expression further comprises using a transfection-facilitating polypeptide, wherein the transfection-facilitating polypeptide comprises a charged polypeptide (e.g. poly-L-glutamate). Increasing GH values are reflected by increasing hematological parameters in the subject having the delivered recombinant dGHRH or functional biological equivalent thereof.

BRIEF DESCRIPTION OF FIGURES

[0024] FIG. 1 shows the alignment of HV-GHRH and dGHRH coding sequences, and the consensus sequence;

[0025] FIG. 2 shows alignment of HV-GHRH and dGHRH amino acid sequences, and the consensus sequence; notice that the 5' signal peptide contains 30 amino acids in the dog specific sequence, and it contains 31 amino acids in the HV-GHRH, a modified porcine GHRH with long serum half-life;

[0026] FIG. 3 shows the sequence of the pAV0221 plasmid vector containing the dGHRH sequence;

[0027] FIG. 4 shows the sequence of the pAV0215 plasmid vector containing the HV-GHRH sequence;

[0028] FIG. 5 shows the average red blood cell count in dogs treated with the species specific dGHRH versus controls and dogs treated with the modified porcine HV-GHRH (day 7, P<0.05 versus baseline in dogs treated with dGHRH);

[0029] FIG. 6 shows the average hemoglobin in dogs treated with the species specific dGHRH versus controls and dogs treated with the modified porcine HV-GHRH (day 14, P<0.05 versus baseline in dogs treated with dGHRH);

[0030] FIG. 7 shows the average mean corpuscular hemoglobin in dogs treated with the species specific dGHRH versus controls and dogs treated with the modified porcine HV-GHRH (day 14, P<0.01 versus baseline in dogs treated with dGHRH); and

[0031] FIG. 8 shows the average mean corpuscular hemoglobin concentration in dogs treated with the species specific dGHRH versus controls and dogs treated with the modified porcine HV-GHRH (day 14, P<0.002 versus baseline in dogs treated with dGHRH).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0032] Terms:

[0033] The term "analog" as used herein includes any mutant of GHRH, or synthetic or naturally occurring peptide fragments of GHRH.

[0034] The terms "canine" and "dog" as used interchangeably herein.

[0035] The term "codon" as used herein refers to any group of three consecutive nucleotide bases in a given messenger RNA molecule, or coding strand of DNA that specifies a particular amino-acid, or a starting or stopping signal for translation. The term codon also refers to base triplets in a DNA strand.

[0036] The term "coding region" as used herein refers to any portion of the DNA sequence that is transcribed into messenger RNA (mRNA) and then translated into a sequence of amino acids characteristic of a specific polypeptide.

[0037] The term "delivery" as used herein is defined as a means of introducing a material into a subject, a cell or any recipient, by means of chemical or biological process, injection, mixing, electroporation, sonoporation, or combination thereof, either without or under pressure.

[0038] The term "encoded GHRH" as used herein is a biologically active polypeptide.

[0039] The term "functional biological equivalent" of GHRH as used herein is a polypeptide that has been engineered to contain a distinct amino acid sequence while simultaneously having similar or improved biological activity when compared to the GHRH polypeptide.

[0040] The term "growth hormone" ("GH") as used herein is defined as a hormone that relates to growth and acts as a chemical messenger to exert its action on a target cell.

[0041] The term "growth hormone releasing hormone" ("GHRH") as used herein is defined as a hormone that facilitates or stimulates release of GH, and to a lesser extent other pituitary hormones, such as prolactin.

[0042] The term "heterologous nucleic acid sequence" as used herein is defined as a DNA sequence consisting of differing regulatory and expression elements.

[0043] The term "isolated" as used herein refers to synthetic or recombinant preparation of molecules in a purified, or concentrated, or both, form, substantially free from undesirable properties.

[0044] The term "modified GHRH" as used herein is a polypeptide that has been engineered to contain an amino acid sequence that is distinct from the wild-type GHRH polypeptide while simultaneously having similar or improved biologically activity when compared to the wild-type GHRH polypeptide. The wild-type GHRH polypeptide is the naturally occurring species-specific GHRH polypeptide of a subject, a cell or any recipient of the modified GHRH.

[0045] The term "nucleic acid expression construct" as used herein refers to any type of genetic construct comprising a nucleic acid coding for a RNA capable of being transcribed. The transcribed RNA is then capable of being translated into a peptide, polypeptide, or protein. The term "expression vector" or "expression plasmid" can also be used interchangeably.

[0046] The term "recipient subject" as used herein refers to a subject that receives a treatment or composition.

[0047] The term "subject" as used herein refers to any species of the animal kingdom, including humans. In preferred embodiments it refers more specifically to canines.

[0048] The term "domesticated animal" as used herein refers to animals used for: pets (e.g. cats, dogs, etc.); work (e.g. horses, cows, etc.); food (chicken, fish, lambs, pigs, etc); and all others known in the art.

[0049] The term "operatively linked" as used herein refers to elements or structures in a nucleic acid sequence that are linked by operative ability and not physical location. The elements or structures are capable of, or characterized by accomplishing a desired operation. It is recognized by one of ordinary skill in the art that it is not necessary for elements or structures in a nucleic acid sequence to be in a tandem or adjacent order to be operatively linked.

[0050] The term "promoter" as used herein refers to a sequence of DNA that directs the transcription of a gene. A promoter may direct the transcription of a prokaryotic or eukaryotic gene. A promoter may be "inducible", initiating transcription in response to an inducing agent or, in contrast, a promoter may be "constitutive", whereby an inducing agent does not regulate the rate of transcription. A promoter may be regulated in a tissue-specific or tissue-preferred manner, such that it is only active in transcribing the operable linked coding region in a specific tissue type or types.

[0051] The term "replication element" as used herein comprises nucleic acid sequences that will lead to replication of a plasmid in a specified host. One skilled in the art of molecular biology will recognize that the replication element may include, but is not limited to, a selectable marker gene promoter, a ribosomal binding site, a selectable marker gene sequence, and an origin of replication.

[0052] The term "therapeutic element" as used herein comprises nucleic acid sequences that will lead to an in vivo expression of an encoded gene product. One skilled in the art of molecular biology will recognize that the therapeutic element may include, but is not limited to a promoter sequence, a poly [A] sequence, or a 3' or 5' UTR.

[0053] The term "vector" as used herein refers to any vehicle that delivers a nucleic acid into a cell or organism. Examples include plasmid vectors, viral vectors, liposomes, or cationic lipids.

[0054] The standard one and three letter abbreviations for amino acids used herein

[0055] are as follows: Alanine, A ala; Arginine, R, arg; Asparagine, N, asn; Aspartic acid, D, asp; Cysteine, C, cys; Glutamine, Q, gln; Glutamic acid, E, glu; Glycine, G, gly; Histidine, H, his; Isoleucine, I, ile; Leucine, L, leu; Lysine, K, lys; Methionine, M, met; Phenylalanine, F, phe; Proline, P, pro; Serine, S, ser; Threonine, T, thr; Tryptophan, W, trp; Tyrosine, Y, tyr; Valine, V, val.

[0056] In a preferred embodiment, the nucleic acid construct or vector of the present invention is a plasmid that comprises a synthetic myogenic (muscle-specific) promoter, a synthetic nucleotide sequence encoding a dGHRH or its analog, and a 3' untranslated region (3'UTR).

[0057] Promoters and Enhancers. A "promoter" is a control sequence that is a region of a nucleic acid sequence at which the initiation and rate of transcription are controlled. It may contain genetic elements where regulatory proteins and molecules may bind such as RNA polymerase and 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.

[0058] 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." Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, 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. Such 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. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR.TM.. Furthermore, it is contemplated the 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.

[0059] Naturally, it will be important to employ a 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. 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. In a specific embodiment the promoter is a synthetic myogenic promoter (Seq. ID No. 11).

[0060] The identity of tissue-specific promoters or elements, as well as assays to characterize their activity, is well known to those of skill in the art. Examples of such regions include the human LIMK2 gene, the somatostatin receptor 2 gene, murine epididymal retinoic acid-binding gene, human CD4, mouse alpha2 (XI) collagen, D1A dopamine receptor gene, insulin-like growth factor II, human platelet endothelial cell adhesion molecule-1.

[0061] Initiation Signals and Internal Ribosome Binding Sites. A specific initiation signal also may be required for efficient translation (synthesis of the encoded protein) 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.

[0062] In certain embodiments of the invention, internal ribosome entry sites ("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. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described, as well a IRES from a mammalian message. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by a IRES, creating polycistronic messages. By virtue of the IRES element, 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.

[0063] Multiple Cloning Sites. 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. "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. Frequently, 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.

[0064] Splicing Sites. Most transcribed eukaryotic 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 of the transcript for protein expression.

[0065] Polyadenylation Signals. In expression, one will typically include a 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 bovine or human GH polyadenylation signal, convenient and/or known to function well in various target cells. In a specific embodiment the polyadenylation signal is a fragment of the 3'UTR of human growth hormone (Seq. ID No. 12). 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.

[0066] Origins of Replication. In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated. In a specific embodiment the origin of replication is the pUC-18 origin of replication (Seq. ID No. 16). Alternatively an autonomously replicating sequence ("ARS") can be employed if the host cell is yeast.

[0067] Selectable and Screenable Markers. In certain embodiments of the invention, the cells that contain the nucleic acid construct of the present invention may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, 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, such as the antibiotic resistance gene on the plasmid constructs (such as kanamycin, ampicylin, gentamycin, tetracycline, or chloramphenicol). initiated. In a specific embodiment the selectable marker is the kanamycin resistance marker (Seq. ID No. 15).

[0068] Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to 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 calorimetric analysis, are also contemplated. Alternatively, screenable enzymes may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable and screenable markers are well known to one of skill in the art.

[0069] GHRH: GHRH has a short half-life in the circulatory system in mammals (Frohman et al., 1984). The HV-GHRH super-analog was presented in the U.S. patent application Ser. No. 10/021,403 filed on Dec. 12, 2001 and titled "Administration of nucleic acid sequence to female animal to enhance growth in offspring" with Schwartz, et al., listed as inventors and U.S. Pat. No. 6,551,996 ("the '996 patent"), issued on Apr. 22, 2003 having Schwartz et al., listed as inventors. The '996 patent teaches application of a GHRH analog containing mutations that improve the ability to elicit the release of GH. In addition, the '996 patent relates to the treatment of growth deficiencies; the improvement of growth performance; the stimulation of production of GH in an animal at a greater level than that associated with normal growth; and the enhancement of growth utilizing the administration of GH releasing hormone analog and is herein incorporated by reference. In order to clone the dGHRH, a dog hypothalamic library was generated and screened.

[0070] The invention may be better understood with reference to the following examples, which are representative of some of the embodiments of the invention, and are not intended to limit the invention.

Example 1

[0071] DNA/Plasmid constructs: GHRH cDNA constructs were introduced into the pAV plasmid backbone, as described in US patent application Ser. No. 60/396,247 filed on Jul. 16, 2002 and titled "Codon Optimized Synthetic Plasmids" with Draghia-Akli, et al., listed as inventors. Each of the expression vector elements were operatively linked and incorporated into the myogenic GHRH expression vectors. For example, the biological potency of the dGHRH was tested using the pAV plasmid vector that was engineered to direct high levels of skeletal muscle-specific gene expression with the use of a synthetic muscle promoter, SPc5-12 (Li et al., 1999) and a 225-bp fragment of dGHRH, which encodes the 30 amino acid signal peptide and a form of the mature peptide dGHRH (Tyr1-Gly40) followed by the 3' untranslated region of the human GH ("hGH"). The sequence of the muscle specific synthetic promoter (Seq. ID No. 11) and the sequence of the fragment of 3'UTR of human growth hormone (Seq. ID No. 12) are included. Other constructs included the modified porcine HV-GHRH, which was used as a positive control, or another functional biological equivalent thereof. The wild type and mutated porcine GHRH cDNAs were generated by site directed mutagenesis of GHRH cDNA (Altered Sites II in vitro Mutagenesis System, Promega, Madison, Wis.), and cloned into the BamHI/Hind III sites of pSPc5-12, to generate pSP-wt-GHRH, or pSP-TI-GHRH respectively. The 3' untranslated region (3'UTR) of GH was cloned downstream of GHRH cDNA. Each of the resultant plasmids contained a coding region for either a wild type or a mutated form of GHRH. Although not wanting to be bound by theory, some of the mutated resultant amino acid sequences were not naturally present in mammals.

[0072] Dog GHRH cloning--A custom cDNA library was constructed by Clontech Laboratories, Inc., Palo Alto, Calif. The starting tissue for the library was dog hypothalamus (4.7 gm) which had been collected from dogs kept in a closed, experimental facility (NIH Regulations) from birth to death and stored at -80.degree. C. The cDNA library was screened by PCR using a 5' primer selected from the Bam/Hind III fragment of HV-GHRH and a 3' primer selected from sequence in Exon 5 of bovine GHRH.

TABLE-US-00001 Seq ID No. 07 Bam/Hind III 5' Primer: ATG GTG CTC TGG GTG TTC TT Seq ID No. 08 Exon 5 3' Primer: TTC ATC CTT GGG AGT TCC TG

PCR conditions were as following: DNA (library) 3 .mu.l, 10.times. Accutaq buffer 5 .mu.l, DMSO 1 .mu.l, dNTP's (10 mM) 1 .mu.l, Exon3-5' primer (50 ng) 1 .mu.l, Exon 5-3'primer (50 ng) 1 .mu.l, water 37.5 .mu.l, Accutaq 0.5 .mu.l, with the following cycling parameters: 94.degree. C. 10 min, 94.degree. C. 30 sec, 55.degree. C. 30 sec, 68.degree. C. 30 sec for 35 cycles, followed by a cycle at 68.degree. C. for 5 min.

[0073] The PCR fragment generated, approx. 200 bp, was subcloned using the TOPO cloning kit and sent for sequencing. Clone # 13 was found to be complete and aligned and compared with other GHRH sequences, as that of human GHRH.

[0074] Primers were designed with specific mutations to incorporate a restriction sites to facilitate sub-cloning into expression vectors: NcoI, Hind III sites and 2 stop codons in clone #13 for insertion into the new pAV backbone. The newly generated expected band size is approx. 240 bp.

TABLE-US-00002 Seq ID No. 09 dogHindIII B 5'Primer CGGCCGAAAGCTTACTATGCTCCT Seq ID No. 10 dogNcoI B 3'Primer ATTCGCCCCCATGGTGCTCTGGG

[0075] PCR Conditions were as following: DNA (clone #13) 10 ng, 10.times. Accutaq buffer 5 .mu.l, DMSO 1 .mu.l, dNTP's (10 mM) 1 .mu.l, 5' primer (50 ng) 1 .mu.l, 3'primer (50 ng) 1 .mu.l, water 40.5 .mu.l, Accutaq 0.5 .mu.l. The cycling parameters were as following: 95.degree. C. for 3' min, 94.degree. C. 30 sec, 52.degree. C. 30 sec, 68.degree. C. 30 sec, for 30 cycles, followed by on extension at 68.degree. C. for 5 min.

[0076] PCR reaction mix digested with NcoI and HindIII and ligated into the new backbone using Takara ligase; clones were then sequenced to confirm that restriction sites and stop codons had been incorporated. Muscle cells (Sol 8's) were transfected with the resulting vector and a Northern blot confirmed presence of species specific RNA.

[0077] An alignment of the HV-GHRH and dGHRH coding sequences is shown in FIG. 1, and an alignment of the corresponding amino acid sequences is shown in FIG. 2. As shown below, the encoded GHRH amino acid sequences are different:

TABLE-US-00003 Porcine (pGHRH): SEQ ID NO. 01 YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGERNQEQGA-OH Mutated porcine (HV-GHRH): SEQ ID NO. 02 HVDAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQGA-OH Canine or Dog specific (dGHRH): SEQ ID NO. 03 YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGERNREQGA-OH

[0078] Although not wanting to be bound by theory, the effects of treating a GH deficient diseases or anemia is determined ultimately by the circulating levels of needed hormones. In general, the encoded dGHRH or functional biological equivalent thereof is of formula:

TABLE-US-00004 SEQ ID NO. 04 X.sub.1X.sub.2DAIFTNSYRKVLX.sub.3QLSARKLLQDIX.sub.4X.sub.5RQQGERNREQGA

wherein: X.sub.1 is a D- or L-isomer of an amino acid selected from the group consisting of tyrosine ("Y"), or histidine ("H"); X.sub.2 is a D- or L-isomer of an amino acid selected from the group consisting of alanine ("A"), valine ("V"), or isoleucine ("I"); X.sub.3 is a D- or L-isomer of an amino acid selected from the group consisting of alanine ("A") or glycine ("G"); X.sub.4 is a D- or L-isomer of an amino acid selected from the group consisting of methionine ("M"), or leucine ("L"); X.sub.5 is a D- or L-isomer of an amino acid selected from the group consisting of serine ("S") or asparagines ("N").

[0079] The pAV0221 plasmid shown in FIG. 3 and Seq ID No. 05 comprises a coding region for the dGHRH. The pAV0215 plasmid shown in FIG. 4 and Seq ID No. 06 comprises a coding region for the mutated HV-GHRH. The plasmids described above do not contain polylinker, IGF-I gene, a skeletal alpha-actin promoter or a skeletal alpha actin 3' UTR/NCR. Furthermore, these plasmids were introduced by muscle injection, followed by in vivo electroporation, as described below.

[0080] In terms of "functional biological equivalents", it is well understood by the skilled artisan that, inherent in the definition of a "biologically functional equivalent" protein and/or polynucleotide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule while retaining a molecule with an acceptable level of equivalent biological activity. Functional biological equivalents are further defined herein as those proteins (and polynucleotides) in selected amino acids (or codons) may be substituted. A peptide comprising a functional biological equivalent of a species specific GHRH is a polypeptide that has been engineered to contain distinct amino acid sequences while simultaneously having similar or improved biologically activity when compared to GHRH. For example one biological activity of GHRH is to facilitate GH secretion in the subject. Another example of a functional biological equivalent is a biologically active peptide or nucleic acid sequence having at least 95% identity to any of the corresponding Seq ID No's.: 1-10.

Example 2

[0081] Experimental animals: Nine dogs were divided into three groups of 3 animals. All dogs were of approximately the same age (.+-.1-2 months), at least 1 year-old, and weight within 5% of each other. Group I received water for injection on day 1 and constitute the negative control group. Group II received 1 mg dog-GHRH plasmid on day 1 and constitute the test group. Group III received 1 mg HV-GHRH plasmid on day 1 and constitute the positive control group. Plasmid formulation or water for injection was administered by intramuscular injection followed by electroporation on Day 1. Blood samples for measurement of hematology, serum chemistry and hormone parameters were collected during physical exams on Day -6 and on Day 1 prior to dosing. Additional blood collections were done every following week to the end of the study. Urine was collected and urinalysis performed on Day 1 prior to dosing and at termination of the study for each animal. Injection sites (medial thigh) were examined for signs of erythema and edema during physical examinations, at dosing on Day 1, Day 2 and every week thereafter. The following hematological parameters were measured at the indicated time points: erythrocyte counts ("RBC"), hematocrit, hemoglobin, total leukocyte count ("WBC"), and differential leukocyte counts (neutrophils, lymphocytes, monocytes, eosinophils, and basophils), platelet count, MCV, MCH, MCHC.

[0082] Red blood cell production. As shown in FIG. 5, the dog group that was treated with the plasmid encoding the dGHRH (.quadrature.) showed an increase (P<0.05) in red blood cell count as soon as day 7 post-injection when compared to baseline values. By comparison, the dogs treated with the plasmid encoding the modified porcine HV-GHRH (.DELTA.) molecule, which was proved to stimulate hematopoiesis long-term post-injection, did not show any improvement in their day 7 hematological parameters.

[0083] Hemoglobin production. As shown in FIG. 6, the Beagle dogs that were injected with the plasmid encoding the dGHRH (.quadrature.) showed an increase in the production of hemoglobin (P<0.05) at 14 days post-injection. Additionally, the mean corpuscular hemoglobin for the Beagle dogs that were injection with the plasmid encoding the dGHRH (.quadrature.) was also increased (P<0.01), as shown in FIG. 7. The mean corpuscular hemoglobin concentration increased when compared to baseline values in the dog group treated with the plasmid encoding dGHRH (P<0.002), as shown in FIG. 8. In contrast, the dogs treated with the plasmid encoding the modified porcine HV-GHRH (.DELTA.) molecule, which was proved to stimulate hematopoiesis long-term post-injection, did not show any improvement in their hematological parameters.

[0084] Although not wanting to be bound by theory, regulatory hormones (e.g. GHRH and GH) often contain a complex feedback-regulated pathway, which are further complicated by chronic conditions (e.g. cancer, immunodeficiency syndromes, and others). Without direct experimentation of the GHRH or biological equivalents that are used in plasmid mediated supplementation, a beneficial therapy could not be predicted by one skilled in the art to determine what modifications to the encoded GHRH or it's functional biological equitant will yield a desired result. For example, previous experiments have indicated that the modified mammalian HV-GHRH produced desired affects faster than the porcine wild-type GHRH (Draghia-Akli et al., 1999). As shown in the example described above, the dGHRH improved canine hematological parameters faster and more efficiently than the modified mammalian HV-GHRH. The invention described herein contains the compositions, descriptions, and results of essential experimentation that explored species specific of distinctive nucleic acid sequences that encoded for a dGHRH or biological equivalent thereof, which was not obvious based upon prior art.

[0085] One skilled in the art readily appreciates that the disclosed invention is well adapted to carry out the mentioned and inherent objectives. GH, GHRH, modified growth hormone releasing hormone or functional biological equivalents, plasmids, vectors, pharmaceutical compositions, treatments, methods, procedures and techniques described herein are presented as representative of the preferred embodiments and are not intended as limitations of the scope of the invention. Thus, other uses will occur to those skilled in the art that are encompassed within the spirit and scope of the described invention.

[0086] The entire content of each of the following documents and publications are hereby incorporated by reference.

TABLE-US-00005 U.S. PATENT DOCUMENTS No. Number Date Inventor 1 U.S. Pat. No. 60/396,247 Jul. 16, 2002 Draghia-Akli 2 U.S. Pat. No. 6,551,996 Apr. 22, 2003 Schwartz 3 U.S. Pat. No. 5,872,127 Feb. 16, 1999 Cincotta 4 U.S. Pat. No. 5,847,066 Dec. 08, 1998 Coy 5 U.S. Pat. No. 5,846,936 Dec. 08, 1998 Felix 6 U.S. Pat. No. 5,792,747 Aug. 11, 1998 Schally 7 U.S. Pat. No. 5,776,901 Jul. 07, 1998 Bowers 8 U.S. Pat. No. 5,756,264 May 26, 1998 Schwartz 9 U.S. Pat. No. 5,704,908 Jan. 06, 1998 Hoffman 10 U.S. Pat. No. 5,702,304 Dec. 30, 1997 Acres, et al. 11 U.S. Pat. No. 5,696,089 Dec. 09, 1997 Felix 12 U.S. Pat. No. 5,605,885 Feb. 25, 1997 Bernton 13 U.S. Pat. No. 5,486,505 Jan. 23, 1996 Bowers 14 U.S. Pat. No. 5,439,440 Aug. 08, 1995 Hoffman 15 U.S. Pat. No. 5,292,721 Mar. 08, 1994 Boyd 16 U.S. Pat. No. 5,137,872 Aug. 11, 1992 Seely 17 U.S. Pat. No. 5,134,120 Jul. 28, 1992 Boyd 18 U.S. Pat. No. 5,084,442 Jan. 28, 1992 Felix 19 U.S. Pat. No. 5,061,690 Oct. 29, 1991 Kann 20 U.S. Pat. No. 5,036,045 Jul. 30, 1991 Thorner 21 U.S. Pat. No. 5,023,322 Jun. 11, 1991 Kovacs 22 U.S. Pat. No. 4,956,288 Sep. 11, 1990 Barsoum 23 U.S. Pat. No. 4,839,344 Jun. 13, 1989 Bowers 24 U.S. Pat. No. 4,410,512 Oct. 18, 1983 Bowers 25 US-RE33,699 Sep. 24, 1991 Drengler 26 U.S. Pat. No. 4,833,166 May 23, 1989 Grosvenor 27 U.S. Pat. No. 4,228,158 Oct. 14, 1980 Momany 28 U.S. Pat. No. 4,228,156 Oct. 14, 1980 Momany 29 U.S. Pat. No. 4,226,857 Oct. 07, 1980 Momany 30 U.S. Pat. No. 4,224,316 Sep. 23, 1980 Momany 31 U.S. Pat. No. 4,223,021 Sep. 16, 1980 Momany 32 U.S. Pat. No. 4,223,020 Sep. 16, 1980 Momany 33 U.S. Pat. No. 4,223,019 Sep. 16, 1980 Momany

OTHER LITERATURE

TABLE-US-00006 [0087] 34 PCT WO 96/12520 35 PCT WO 96/12006 36 PCT WO 95/19805 37 PCT WO 97/07826

REFERENCE LIST

[0088] Acsadi, G., G. Dickson, D. R. Love, A. Jani, F. S. Walsh, A. Gurusinghe, Wolff, J A, and K. E. Davies. 1991. Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs. Nature 352:815-818. [0089] Aihara, H. and J. Miyazaki. 1998. Gene transfer into muscle by electroporation in vivo. Nat. Biotechnol. 16:867-870. [0090] Aratani, Y., R. Okazaki, and H. Koyama. 1992. End extension repair of introduced targeting vectors mediated by homologous recombination in mammalian cells. Nucleic Acids Res. 20:4795-4801. [0091] Bettan, M., F. Emmanuel, R. Darteil, J. M. Caillaud, F. Soubrier, P. Delaere, D. Branelec, A. Mahfoudi, N. Duverger, and D. Scherman. 2000. High-level protein secretion into blood circulation after electric pulse-mediated gene transfer into skeletal muscle. Mol. Ther. 2:204-210. [0092] Butler, A. A., G. R. Ambler, B. H. Breier, D. LeRoith, C. T. Roberts, Jr., and P. D. Gluckman. 1994. Growth hormone (GH) and insulin-like growth factor-I (IGF-I) treatment of the GH-deficient dwarf rat: differential effects on IGF-I transcription start site expression in hepatic and extrahepatic tissues and lack of effect on type I IGF receptor mRNA expression. Mol. Cell Endocrinol. 101:321-330. [0093] Caroni, P. and C. Schneider. 1994. Signaling by insulin-like growth factors in paralyzed skeletal muscle: rapid induction of IGF 1 expression in muscle fibers and prevention of interstitial cell proliferation by IGF-BP5 and IGF-BP4. J. Neurosci. 14:3378-3388. [0094] Corpas, E., S. M. Harman, M. A. Pineyro, R. Roberson, and M. R. Blackman. 1993. Continuous subcutaneous infusions of growth hormone (GH) releasing hormone 1-44 for 14 days increase GH and insulin-like growth factor-I levels in old men. Journal of Clinical Endocrinology & Metabolism 76:134-138. [0095] Danko, I. and J. A. Wolff. 1994. Direct gene transfer into muscle. Vaccine 12:1499-1502. [0096] Davis, H. L., R. G. Whalen, and B. A. Demeneix. 1993. Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency oftransfer and stability of expression. Human Gene Therapy 4:151-159. [0097] Dolnik, V., M. Novotny, and J. Chmelik. 1993. Electromigration behavior of poly-(L-glutamate) conformers in concentrated polyacrylamide gels. Biopolymers 33: 1299-1306. [0098] Draghia-Akli, R., K. K. Cummings, A. S. Khan, P. A. Brown, and R. H. Carpenter. 2003a. Effects of plasmid-mediated growth hormone releasing hormone supplementation in young healthy Beagle dogs. Journal of Animal Science 81:2301-2310. [0099] Draghia-Akli, R., K. M. Ellis, L. A. Hill, P. B. Malone, and M. L. Fiorotto. 2003b. High-efficiency growth hormone releasing hormone plasmid vector administration into skeletal muscle mediated by electroporation in pigs. FASEB J 17:526-528. [0100] Draghia-Akli, R., M. L. Fiorotto, L. A. Hill, P. B. Malone, D. R. Deaver, and R. J. Schwartz. 1999. Myogenic expression of an injectable protease-resistant growth hormone-releasing hormone augments long-term growth in pigs. Nat. Biotechnol. 17:1179-1183. [0101] Draghia-Akli, R., K. A. Hahn, G. K. King, K. Cummings, and R. H. Carpenter. 2002a. Effects Of Plasmid Mediated Growth Hormone Releasing Hormone In Severely Debilitated Dogs With Cancer. Molecular Therapy 6:830-836. [0102] Draghia-Akli, R., A. S. Khan, K. K. Cummings, D. Parghi, R. H. Carpenter, and P. A. Brown. 2002b. Electrical Enhancement of Formulated Plasmid Delivery in Animals. Technology in Cancer Research & Treatment 1:365-371. [0103] Draghia-Akli, R., X. G. Li, and R. J. Schwartz. 1997. Enhanced growth by ectopic expression of growth hormone releasing hormone using an injectable myogenic vector. Nat. Biotechnol. 15:1285-1289. [0104] Draghia-Akli, R., P. B. Malone, L. A. Hill, K. M. Ellis, R. J. Schwartz, and J. L. Nordstrom. 2002c. Enhanced animal growth via ligand-regulated GHRH myogenic-injectable vectors. FASEB J. 16:426-428. [0105] Dubreuil, P., D. Petitclerc, G. Pelletier, P. Gaudreau, C. Farmer, Mowles, T F, and P. Brazeau. 1990. Effect of dose and frequency of administration of a potent analog of human growth hormone-releasing factor on hormone secretion and growth in pigs. Journal of Animal Science 68:1254-1268. [0106] Etherton, T. D., J. P. Wiggins, C. S. Chung, C. M. Evock, J. F. Rebhun, and P. E. Walton. 1986. Stimulation of pig growth performance by porcine growth hormone and growth hormone-releasing factor. Journal of Animal Science 63:1389-1399. [0107] Fewell, J. G., F. MacLaughlin, V. Mehta, M. Gondo, F. Nicol, E. Wilson, and L. C. Smith. 2001. Gene therapy for the treatment of hemophilia B using PINC-formulated plasmid delivered to muscle with electroporation. Mol. Ther. 3:574-583. [0108] Foncea, R., M. Andersson, A. Ketterman, V. Blakesley, M. Sapag-Hagar, P. H. Sugden, D. LeRoith, and S. Lavandero. 1997. Insulin-like growth factor-I rapidly activates multiple signal transduction pathways in cultured rat cardiac myocytes. J. Biol. Chem. 272:19115-19124. [0109] Frohman, L. A., T. R. Downs, and P. Chomczynski. 1992. Regulation of growth hormone secretion. Frontiers in Neuroendocrinology 13:344-405. [0110] Frohman, L. A., J. L. Thominet, C. B. Webb, M. L. Vance, H. Uderman, J. Rivier, W. Vale, and M. O. Thorner. 1984. Metabolic clearance and plasma disappearance rates of human pancreatic tumor growth hormone releasing factor in man. J. Clin. Invest. 73:1304-1311. [0111] Fryer, A. D. and D. B. Jacoby. 1993. Effect of inflammatory cell mediators on M2 muscarinic receptors in the lungs. Life Sci. 52:529-536. [0112] Gehl, J., T. Skovsgaard, and L. M. Mir. 1998. Enhancement of cytotoxicity by electropermeabilization: an improved method for screening drugs. Anticancer Drugs 9:319-325. [0113] Gehl, J., T. H. Sorensen, K. Nielsen, P. Raskmark, S. L. Nielsen, T. Skovsgaard, and L. M. Mir. 1999. In vivo electroporation of skeletal muscle: threshold, efficacy and relation to electric field distribution. Biochim. Biophys. Acta 1428:233-240. [0114] Gesundheit, N. and J. K. Alexander. 1995. Endocrine Therapy with Recombinant Hormones and Growth Factors. In: B. D. Weintraub (Ed.) Molecular Endocrinology: Basic Concepts and Clinical Correlations. pp. 491-507. Raven Press Ltd., New York. [0115] Heller, R., M. J. Jaroszeski, L. F. Glass, J. L. Messina, D. P. Rapaport, R. C. DeConti, N. A. Fenske, R. A. Gilbert, L. M. Mir, and D. S. Reintgen. 1996. Phase I/II trial for the treatment of cutaneous and subcutaneous tumors using electrochemotherapy. Cancer 77:964-971. [0116] Hoess, R. H. and K. Abremski. 1985. Mechanism of strand cleavage and exchange in the Cre-lox site-specific recombination system. J. Mol. Biol. 181:351-362. [0117] Kooistra, H. S., G. Voorhout, J. A. Mol, and A. Rijnberk. 2000. Combined pituitary hormone deficiency in german shepherd dogs with dwarfism. Domest. Anim Endocrinol. 19: 177-190. [0118] Kooistra, H. S., G. Voorhout, P. J. Selman, and A. Rijnberk. 1998. Progestin-induced growth hormone (GH) production in the treatment of dogs with congenital GH deficiency. Domest. Anim Endocrinol. 15:93-102. [0119] Lapierre, H., G. Pelletier, D. Petitclerc, P. Dubreuil, J. Morisset, P. Gaudreau, Y. Couture, and P. Brazeau. 1991. Effect of human growth hormone-releasing factor and(or) thyrotropin-releasing factor on growth, carcass composition, diet digestibility, nutrient balance, and plasma constituents in dairy calves. Journal of Animal Science 69:587-598. [0120] Lesbordes, J. C., T. Bordet, G. Haase, L. Castelnau-Ptakhine, S. Rouhani, H. Gilgenkrantz, and A. Kahn. 2002. In vivo electrotransfer of the cardiotrophin-1 gene into skeletal muscle slows down progression of motor neuron degeneration in pmn mice. Hum. Mol. Genet. 11: 1615-1625.

[0121] Li, C., S. Ke, Q. P. Wu, W. Tansey, N. Hunter, L. M. Buchmiller, L. Milas, C. Charnsangavej, and S. Wallace. 2000. Tumor irradiation enhances the tumor-specific distribution of poly(L-glutamic acid)-conjugated paclitaxel and its antitumor efficacy. Clin. Cancer Res. 6:2829-2834. [0122] Li, X., E. M. Eastman, R. J. Schwartz, and R. Draghia-Akli. 1999. Synthetic muscle promoters: activities exceeding naturally occurring regulatory sequences. Nat. Biotechnol. 17:241-245. [0123] Liu, J. L. and D. LeRoith. 1999. Insulin-like growth factor I is essential for postnatal growth in response to growth hormone. Endocrinology 140:5178-5184. [0124] Lowe, W. L., Jr., M. Adamo, H. Werner, C. T. Roberts, Jr., and D. LeRoith. 1989. Regulation by fasting of rat insulin-like growth factor I and its receptor. Effects on gene expression and binding. J. Clin. Invest 84:619-626. [0125] Lucas, M. L., L. Heller, D. Coppola, and R. Heller. 2002. IL-12 plasmid delivery by in vivo electroporation for the successful treatment of established subcutaneous B16.F10 melanoma. Mol. Ther. 5:668-675. [0126] Lucas, M. L., M. J. Jaroszeski, R. Gilbert, and R. Heller. 2001. In vivo electroporation using an exponentially enhanced pulse: a new waveform. DNA Cell Biol. 20:183-188. [0127] Matsubara, H., Y. Gunji, T. Maeda, K. Tasaki, Y. Koide, T. Asano, T. Ochiai, S. Sakiyama, and M. Tagawa. 2001. Electroporation-mediated transfer of cytokine genes into human esophageal tumors produces anti-tumor effects in mice. Anticancer Res. 21:2501-2503. [0128] Matsuo, A., I. Tooyama, S. Isobe, Y. Oomura, I. Akiguchi, K. Hanai, J. Kimura, and H. Kimura. 1994. Immunohistochemical localization in the rat brain of an epitope corresponding to the fibroblast growth factor receptor-1. Neuroscience 60:49-66. [0129] McNally, M. A., J. S. Lebkowski, T. B. Okarma, and L. B. Lerch. 1988. Optimizing electroporation parameters for a variety of human hematopoietic cell lines. Biotechniques 6:882-886. [0130] Miklavcic, D., K. Beravs, D. Semrov, M. Cemazar, F. Demsar, and G. Sersa. 1998. The importance of electric field distribution for effective in vivo electroporation of tissues. Biophys. J 74:2152-2158. [0131] Mumper, R. J., J. Wang, S. L. Klakamp, H. Nitta, K. Anwer, F. Tagliaferri, and A. P. Rolland. 1998. Protective interactive noncondensing (PINC) polymers for enhanced plasmid distribution and expression in rat skeletal muscle. J. Control Release 52: 191-203. [0132] Muramatsu, T., S. Arakawa, K. Fukazawa, Y. Fujiwara, T. Yoshida, R. Sasaki, S. Masuda, and H. M. Park. 2001. In vivo gene electroporation in skeletal muscle with special reference to the duration of gene expression. Int. J. Mol. Med. 7:37-42. [0133] Nairn, R. S., G. M. Adair, T. Porter, S. L. Pennington, D. G. Smith, J. H. Wilson, and M. M. Seidman. 1993. Targeting vector configuration and method of gene transfer influence targeted correction of the APRT gene in Chinese hamster ovary cells. Somat. Cell Mol. Genet. 19:363-375. [0134] Neumann, E., M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider. 1982. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J. 1:841-845. [0135] Otani, Y., Y. Tabata, and Y. Ikada. 1996. Rapidly curable biological glue composed of gelatin and poly(L-glutamic acid). Biomaterials 17:1387-1391. [0136] Otani, Y., Y. Tabata, and Y. Ikada. 1998. Hemostatic capability of rapidly curable glues from gelatin, poly(L-glutamic acid), and carbodiimide. Biomaterials 19:2091-2098. [0137] Parks, J. S., R. W. Pfaffle, M. R. Brown, H. Abdul-Latif, and L. R. Meacham. 1995. Growth Hormone Deficiency. In: B. D. Weintraub (Ed.) Molecular Endocrinology: Basic Concepts and Clinical Correlations. pp. 473-490. Raven Press, Ltd., New York. [0138] Parrizas, M. and D. LeRoith. 1997. Insulin-like growth factor-1 inhibition of apoptosis is associated with increased expression of the bcl-xL gene product. Endocrinology 138: 1355-1358. [0139] Rabinovsky, E. D., G. M. Smith, D. P. Browder, H. D. Shine, and J. L. McManaman. 1992. Peripheral nerve injury down-regulates CNTF expression in adult rat sciatic nerves. J. Neurosci. Res. 31:188-192. [0140] Rijnberk, A., H. van Herpen, J. A. Mol, and G. R. Rutteman. 1993. Disturbed release of growth hormone in mature dogs: a comparison with congenital growth hormone deficiency. Vet. Rec. 133:542-545. [0141] Smith, L. C. and J. L. Nordstrom. 2000. Advances in plasmid gene delivery and expression in skeletal muscle. Curr. Opin. Mol. Ther. 2:150-154. [0142] Terada, Y., H. Tanaka, T. Okado, S. Inoshita, M. Kuwahara, T. Akiba, S. Sasaki, and F. Marumo. regulated erythropoietin production by naked dna injection and in vivo electroporation. Am. J Kidney Dis. 38:S50-S53. [0143] Thorner, M. O., L. A. Frohman, D. A. Leong, J. Thominet, T. Downs, P. Hellmann, J. Chitwood, J. M. Vaughan, and W. Vale. 1984. Extrahypothalamic growth-hormone-releasing factor (GRF) secretion is a rare cause of acromegaly: plasma GRF levels in 177 acromegalic patients. Journal of Clinical Endocrinology & Metabolism 59:846-849. [0144] Toneguzzo, F., A. Keating, S. Glynn, and K. McDonald. 1988. Electric field-mediated gene transfer: characterization of DNA transfer and patterns of integration in lymphoid cells. Nucleic Acids Res. 16:5515-5532. [0145] Tripathy, S. K., E. C. Svensson, H. B. Black, E. Goldwasser, M. Margalith, Hobart, P M, and J. M. Leiden. 1996. Long-term expression of erythropoietin in the systemic circulation of mice after intramuscular injection of a plasmid DNA vector. Proc. Natl. Acad. Sci. USA 93:10876-10880. [0146] Tsurumi, Y., S. Takeshita, D. Chen, M. Kearney, S. T. Rossow, J. Passeri, J. R. Horowitz, J. F. Symes, and J. M. Isner. 1996. Direct intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor augments collateral development and tissue perfusion [see comments]. Circulation 94:3281-3290. [0147] van Rooij, E. M., B. L. Haagmans, H. L. Glansbeek, Y. E. de Visser, M. G. de Bruin, W. Boersma, and A. T. Bianchi. 2000. A DNA vaccine coding for glycoprotein B of pseudorabies virus induces cell-mediated immunity in pigs and reduces virus excretion early after infection. Vet. Immunol. Immunopathol. 74:121-136. [0148] Vance, M. L. 1990. Growth-hormone-releasing hormone. [Review] [52 refs]. Clinical Chemistry 36:415-420. [0149] Vance, M. L., D. L. Kaiser, W. S. Evans, R. Furlanetto, W. Vale, J. Rivier, and M. O. Thorner. 1985. Pulsatile growth hormone secretion in normal man during a continuous 24-hour infusion of human growth hormone releasing factor (1-40). Evidence for intermittent somatostatin secretion. J. Clin. Invest. 75:1584-1590. [0150] Veldhuis, J. D., A. Iranmanesh, and A. Weltman. 1997. ELEMENTS IN THE PATHOPHYSIOLOGY OF DIMINISHED GROWTH HORMONE (GH) SECRETION IN AGING HUMANS. Endocrine 7:41-48. [0151] Vilquin, J. T., P. F. Kennel, M. Patumeau-Jouas, P. Chapdelaine, N. Boissel, P. Delaere, J. P. Tremblay, D. Scherman, M. Y. Fiszman, and K. Schwartz. 2001. Electrotransfer of naked DNA in the skeletal muscles of animal models of muscular dystrophies. Gene Ther. 8:1097-1107. [0152] Wolff, J. A., R. W. Malone, P. Williams, W. Chong, G. Acsadi, A. Jani, Felgner, and PL. 1990. Direct gene transfer into mouse muscle in vivo. Science 247: 1465-1468. [0153] Xie, T. D. and T. Y. Tsong. 1993. Study of mechanisms of electric field-induced DNA transfection. V. Effects of DNA topology on surface binding, cell uptake, expression, and integration into host chromosomes of DNA in the mammalian cell. Biophys. J. 65:1684-1689. [0154] Yasui, A., K. Oda, H. Usunomiya, K. Kakudo, T. Suzuki, T. Yoshida, H. M. Park, K. Fukazawa, and T. Muramatsu. 2001. Elevated gastrin secretion by in vivo gene electroporation in skeletal muscle. Int. J. Mol. Med. 8:489-494. [0155] Yin, D. and J. G. Tang. 2001. Gene therapy for streptozotocin-induced diabetic mice by electroporational transfer of naked human insulin precursor DNA into skeletal muscle in vivo. FEBS Lett. 495:16-20. [0156] Yorifuji, T. and H. Mikawa. 1990. Co-transfer of restriction endonucleases and plasmid DNA into mammalian cells by electroporation: effects on stable transformation. Mutat. Res. 243:121-126.

Sequence CWU 1

1

22140PRTArtificial sequenceThis is the amino acid sequence for porcine GHRH. 1Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly20 25 30Glu Arg Asn Gln Glu Gln Gly Ala35 40240PRTartificial sequenceThis is a modified amino acid sequence for GHRH 2His Val Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Ala Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Leu Asn Arg Gln Gln Gly20 25 30Glu Arg Asn Gln Glu Gln Gly Ala35 40340PRTartificial sequenceThis is the amino acid sequence for canine GHRH. 3Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly20 25 30Glu Arg Asn Arg Glu Gln Gly Ala35 40440PRTartificial sequenceThis is the general amino acid sequence for canine GHRH. 4Xaa Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Xaa Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Xaa Xaa Arg Gln Gln Gly20 25 30Glu Arg Asn Arg Glu Gln Gly Ala35 4052716DNAArtificial SequencepAV0221 is an expression plasmid having a dGHRH sequence. 5ccaccgcggt ggcggccgtc cgccctcggc accatcctca cgacacccaa atatggcgac 60gggtgaggaa tggtggggag ttatttttag agcggtgagg aaggtgggca ggcagcaggt 120gttggcgctc taaaaataac tcccgggagt tatttttaga gcggaggaat ggtggacacc 180caaatatggc gacggttcct cacccgtcgc catatttggg tgtccgccct cggccggggc 240cgcattcctg ggggccgggc ggtgctcccg cccgcctcga taaaaggctc cggggccggc 300ggcggcccac gagctacccg gaggagcggg aggcgccaag cggatcccaa ggcccaactc 360cccgaaccac tcagggtcct gtggacagct cacctagctg ccatggtgct ctgggtgttc 420ttcctggtga tcctcaccct cagcagtggt tcccactctt ccccgccatc cctgcccatc 480agaatccctc ggtatgcaga cgccatcttc accaacagct accggaaggt gctgggccag 540ctgtccgccc gcaagctcct scaggacatc atgagccggc agcagggaga gagaaaccgg 600gagcaaggag catagtaagc ttatcggggt ggcatccctg tgacccctcc ccagtgcctc 660tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt 720gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat 780ggagcaaggg gcaagttggg aagacaacct gtagggctcg agggggggcc cggtaccagc 840ttttgttccc tttagtgagg gttaatttcg agcttggtct tccgcttcct cgctcactga 900ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 960acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 1020aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 1080tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 1140aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 1200gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 1260acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 1320accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 1380ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 1440gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 1500aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 1560ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 1620gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 1680cgctcagcta gcgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa 1740tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct 1800tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg 1860ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca 1920tcgccatgag tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac 1980agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg 2040gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag 2100gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg 2160gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag 2220tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc 2280agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc 2340ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag 2400ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa 2460cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca 2520gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca gtttactttg 2580cagggcttcc caaccttacc agagggcgcc ccagctggca attccggttc gcttgctgtc 2640cataaaaccg cccagtctag caactgttgg gaagggcgat cgtgtaatac gactcactat 2700agggcgaatt ggagct 271662739DNAArtificial SequencepAV0215 is an expression plasmid for HV-GHRH. 6ccaccgcggt ggcggccgtc cgccctcggc accatcctca cgacacccaa atatggcgac 60gggtgaggaa tggtggggag ttatttttag agcggtgagg aaggtgggca ggcagcaggt 120gttggcgctc taaaaataac tcccgggagt tatttttaga gcggaggaat ggtggacacc 180caaatatggc gacggttcct cacccgtcgc catatttggg tgtccgccct cggccggggc 240cgcattcctg ggggccgggc ggtgctcccg cccgcctcga taaaaggctc cggggccggc 300ggcggcccac gagctacccg gaggagcggg aggcgccaag cggatcccaa ggcccaactc 360cccgaaccac tcagggtcct gtggacagct cacctagctg ccatggtgct ctgggtgttc 420ttctttgtga tcctcaccct cagcaacagc tcccactgct ccccacctcc ccctttgacc 480ctcaggatgc ggcggcacgt agatgccatc ttcaccaaca gctaccggaa ggtgctggcc 540cagctgtccg cccgcaagct gctccaggac atcctgaaca ggcagcaggg agagaggaac 600caagagcaag gagcataatg actgcaggaa ttcgatatca agcttatcgg ggtggcatcc 660ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag 720ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat 780tatggggtgg aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc 840tcgagggggg gcccggtacc agcttttgtt ccctttagtg agggttaatt tcgagcttgg 900tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 960tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 1020aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 1080tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 1140tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 1200cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 1260agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 1320tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 1380aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 1440ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 1500cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 1560accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 1620ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 1680ttgatctttt ctacggggtc tgacgctcag ctagcgctca gaagaactcg tcaagaaggc 1740gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt 1800cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat 1860agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca 1920ccatgatatt cggcaagcag gcatcgccat gagtcacgac gagatcctcg ccgtcgggca 1980tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca 2040gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt 2100tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat 2160cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg 2220gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg 2280cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc tgcagttcat 2340tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc 2400ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc 2460tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg 2520atcctcatcc tgtctcttga tcagatcttg atcccctgcg ccatcagatc cttggcggca 2580agaaagccat ccagtttact ttgcagggct tcccaacctt accagagggc gccccagctg 2640gcaattccgg ttcgcttgct gtccataaaa ccgcccagtc tagcaactgt tgggaagggc 2700gatcgtgtaa tacgactcac tatagggcga attggagct 2739720DNAartificial sequenceA 5' primer selected from the Bam/Hind III fragment of HV-GHRH. 7atggtgctct gggtgttctt 20820DNAartificial sequenceA 3' primer selected from sequence in Exon 5 of bovine GHRH. 8ttcatccttg ggagttcctg 20924DNAartificial sequencea Primer designed with specific mutations 9cggccgaaag cttactatgc tcct 241023DNAartificial sequencea Primer designed with specific mutations 10attcgccccc atggtgctct ggg 2311323DNAartificial sequenceNucleic acid sequence of a synthetic eukaryotic promoter SPc5-12 11cggccgtccg ccctcggcac catcctcacg acacccaaat atggcgacgg gtgaggaatg 60gtggggagtt atttttagag cggtgaggaa ggtgggcagg cagcaggtgt tggcgctcta 120aaaataactc ccgggagtta tttttagagc ggaggaatgg tggacaccca aatatggcga 180cggttcctca cccgtcgcca tatttgggtg tccgccctcg gccggggccg cattcctggg 240ggccgggcgg tgctcccgcc cgcctcgata aaaggctccg gggccggcgg cggcccacga 300gctacccgga ggagcgggag gcg 32312190DNAartificial sequenceNucleic acid sequence of a human growth hormone poly A tail. 12gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca 60gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc 120ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca 180acctgtaggg 1901329DNAArtificial SequenceProkaryotic selectable marker gene promoter PNEO. 13accttaccag agggcgcccc agctggcaa 29145DNAartificial sequenceNEO ribosomal binding site. 14tcctc 515795DNAartificial sequenceSequence for antibiotic resistance gene kanamycin. 15atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac 540ggcgaggatc tcgtcgtgac tcatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780gacgagttct tctga 79516782DNAartificial sequenceSequence of a plasmid pUC-18 origin of replication. 16tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 60tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 120aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 180tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 240tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 300cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 360agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 420tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 480aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 540ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 600cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 660accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 720ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 780tt 78217216DNAartificial sequenceThis is the nucleotide sequence of dog GHRH. 17atggtgctct gggtgttctt cctggtgatc ctcaccctca gcagtggttc ccactcttcc 60ccgccatccc tgcccatcag aatccctcgg tatgcagacg ccatcttcac caacagctac 120cggaaggtgc tgggccagct gtccgcccgc aagctcctsc aggacatcat gagccggcag 180cagggagaga gaaaccggga gcaaggagca tagtaa 21618219DNAartificial sequenceThis is the nucleotide sequence of HV-pGHRH. 18atggtgctct gggtgttctt ctttgtgatc ctcaccctca gcaacagctc ccactgctcc 60ccacctcccc ctttgaccct caggatgcgg cggcacgtag atgccatctt caccaacagc 120taccggaagg tgctggccca gctgtccgcc cgcaagctgc tccaggacat cctgaacagg 180cagcagggag agaggaacca agagcaagga gcataatga 21919219DNAartificial sequenceThis is the consensus sequence between dog GHRH and HV-pGHRH. 19atggtgctct gggtgttctt cntngtgatc ctcaccctca gcannngntc ccactnntcc 60ccnccnnccc nnntgnccnt cagnatncnn cggnangnag angccatctt caccaacagc 120taccggaagg tgctggncca gctgtccgcc cgcaagctnc tncaggacat cntgancngg 180cagcagggag agagnaaccn ngagcaagga gcatantna 2192070PRTartificial sequenceThis is the amino acid sequence of dog GHRH. 20Met Val Leu Trp Val Phe Phe Leu Val Ile Leu Thr Leu Ser Ser Gly1 5 10 15Ser His Ser Ser Pro Pro Ser Leu Pro Ile Arg Ile Pro Arg Tyr Ala20 25 30Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln Leu Ser35 40 45Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly Glu Arg50 55 60Asn Arg Glu Gln Gly Ala65 702171PRTartificial sequenceThis is the amino acid sequence of HV-pGHRH. 21Met Val Leu Trp Val Phe Phe Phe Val Ile Leu Thr Leu Ser Asn Ser1 5 10 15Ser His Cys Ser Pro Pro Pro Pro Leu Thr Leu Arg Met Arg Arg His20 25 30Val Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Ala Gln Leu35 40 45Ser Ala Arg Lys Leu Leu Gln Asp Ile Leu Asn Arg Gln Gln Gly Glu50 55 60Arg Asn Gln Glu Gln Gly Ala65 702271PRTartificial sequenceThis is the amino acid consensus sequence between dog GHRH and HV-pGHRH. 22Met Val Leu Trp Val Phe Phe Xaa Val Ile Leu Thr Leu Ser Xaa Xaa1 5 10 15Ser His Xaa Ser Pro Pro Xaa Xaa Leu Xaa Ile Arg Ile Xaa Arg His20 25 30Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Ala Gln Leu35 40 45Ser Ala Arg Lys Leu Leu Gln Asp Ile Leu Xaa Arg Gln Gln Gly Glu50 55 60Arg Asn Xaa Glu Gln Gly Ala65 70

Claims

1. A composition comprising a nucleic acid expression construct encoding a canine specific growth hormone releasing hormone ("dGHRH") or fragments thereof, having a sequence at least 95% identical to SEQ ID NO:4: TABLE-US-00007 X.sub.1X.sub.2DAIFTNSYRKVLX.sub.3QLSARKLLQDIX.sub.4X.sub.5RQQGERNREQGA

wherein: X1 is a D- or L-isomer of an amino acid selected from the group consisting of tyrosine ("Y"), or histidine ("H"); X.sub.2 is a D- or L-isomer of an amino acid selected from the group consisting of alanine ("A"), valine ("V"), or isoleucine ("I"); X.sub.3 is a D- or L-isomer of an amino acid selected from the group consisting of alanine ("A") or glycine ("G"); X.sub.4 is a D- or L-isomer of an amino acid selected from the group consisting of methionine ("M"), or leucine ("L"); X.sub.5 is a D- or L-isomer of an amino acid selected from the group consisting of serine ("S") or asparagines ("N").

2. The composition of claim 1, wherein the dGHRH increases growth hormone ("GH") in a recipient subject.

3. The composition of claim 1, wherein the dGHRH improves hematological parameters in a recipient subject.

4. The composition of claim 1, wherein the dGHRH increases red blood cell values in a recipient subject.

5. The composition of claim 1, wherein the dGHRH increases hemoglobin values in a recipient subject.

6. The composition of claim 1, wherein the dGHRH increases mean corpuscular hemoglobin values in a recipient subject.

7. The composition of claim 1, wherein the nucleic acid expression construct further comprises: (a) a synthetic or eukaryotic promoter; (b) a poly-adenylation signal; (c) a selectable marker gene promoter; (d) a ribosomal binding site; (e) a selectable marker gene sequence; and (f) an origin of replication; wherein, the synthetic or eukaryotic promoter, the nucleic acid sequence encoding the dGHRH or functional biological equivalent thereof, and the poly adenylation signal comprise therapeutic elements of the nucleic acid expression construct; the therapeutic elements are operatively linked and located in a first operatively-linked arrangement; the selectable marker gene promoter, the ribosomal binding site, the selectable marker gene sequence, and the origin of replication comprise replication elements of the nucleic acid expression construct; the replication elements are operatively linked and located in a second operatively-linked arrangement; the first-operatively-linked arrangement and the second-operatively-linked arrangement comprise a circular structure of the nucleic acid expression construct; and the nucleic acid expression construct is utilized for plasmid mediated gene supplementation.

8. The composition of claim 7, further comprising a 5' untranslated region ("UTR") operatively linked to the first operatively-linked arrangement.

9. The composition of claim 7, wherein the 3' untranslated region ("UTR") comprises a portion of a human growth hormone 3'UTR.

10. A nucleic acid expression construct pAV0221 comprising a sequence at least 95% identical to SEQ ID D NO: 5.

11. A nucleic acid expression construct pAV00215 comprising a sequence at least 95% identical to SEQ ID NO: 6.

12. A method of increasing growth hormone ("GH") values in a subject comprising delivering to the subject an isolated composition of canine specific growth hormone releasing hormone ("dGHRH") or fragments thereof, having a sequence at least 95% identical to SEQ ID NO: 4: TABLE-US-00008 X.sub.1X.sub.2DAIFTNSYRKVLX.sub.3QLSARKLLQDIX.sub.4X.sub.5RQQGERNREQGA

wherein: X1 is a D- or L-isomer of an amino acid selected from the group consisting of tyrosine ("Y"), or histidine ("H"); X2 is a D- or L-isomer of an amino acid selected from the group consisting of alanine ("A"), valine ("V"), or isoleucine ("I"); X3 is a D- or L-isomer of an amino acid selected from the group consisting of alanine ("A") or glycine ("G"); X4 is a D- or L-isomer of an amino acid selected from the group consisting of methionine ("M"), or leucine ("L"); X5 is a D- or L-isomer of an amino acid selected from the group consisting of serine ("S") or asparagines ("N").

13. The method of claim 12, wherein the recombinant dGHRH is a biologically active polypeptide, and the recombinant functional biological equivalent of dGHRH is a polypeptide that has been engineered to contain a distinct amino acid sequence while simultaneously having similar or improved biologically activity when compared to the dGHRH polypeptide.

14. The method of claim 12, wherein increasing hematological parameters in the subject having the delivered recombinant dGHRH nucleic acid expression construct reflects increasing GH values.

15. The method of claim 14, wherein the hematological parameter is red blood cell count, hemoglobin concentration, or mean corpuscular hemoglobin.

16. A method of increasing growth hormone ("GH") values in a subject comprising: delivering into cells of the subject a nucleic acid expression construct that encodes a canine specific growth-hormone-releasing-hormone ("dGHRH") or fragments thereof, having a sequence at least 95% identical to SEQ ID NO: 5: TABLE-US-00009 X.sub.1X.sub.2DAIFTNSYRKVLX.sub.3QLSARKLLQDIX.sub.4X.sub.5RQQGERNREQGA

wherein: X1 is a D- or L-isomer of an amino acid selected from the group consisting of tyrosine ("Y"), or histidine ("H"); X2 is a D- or L-isomer of an amino acid selected from the group consisting of alanine ("A"), valine ("V"), or isoleucine ("I"); X3 is a D- or L-isomer of an amino acid selected from the group consisting of alanine ("A") or glycine ("G"); X4 is a D- or L-isomer of an amino acid selected from the group consisting of methionine ("M"), or leucine ("L"); X5 is a D- or L-isomer of an amino acid selected from the group consisting of serine ("S") or asparagines ("N").

17. The method of claim 16, wherein delivering into the cells of the subject the nucleic acid expression construct is via electroporation.

18. The method of claim 16, wherein the cells of the subject are somatic cells, stem cells, or germ cells.

19. The method of claim 16, wherein the nucleic acid expression construct is SEQ ID NO: 5 or SEQ ID NO: 6.

20. The method of claim 16, wherein the nucleic acid expression construct further comprises a transfection-facilitating polypeptide.

21. The method of claim 20, wherein the transfection-facilitating polypeptide comprises a charged polypeptide.

22. The method of claim 20, wherein the transfection-facilitating polypeptide comprises poly-L-glutamate.

23. The method of claim 16, wherein the dGHRH is expressed in tissue-specific cells of the subject.

24. The method of claim 23, wherein the tissue specific cells of the subject comprises muscle cells.

25. The method of claim 16, wherein increasing hematological parameters in the subject having the delivered nucleic acid expression construct reflects increasing GH values.

26. The method of claim 25, wherein the hematological parameter is red blood cell count, hemoglobin concentration, or mean corpuscular hemoglobin.

27. A composition comprising an isolated canine specific growth hormone releasing hormone ("dGHRH") or fragments thereof, having a sequence at least 95% identical to SEQ ID NO: 4: TABLE-US-00010 X.sub.1X.sub.2DAIFTNSYRKVLX.sub.3QLSARKLLQDIX.sub.4X.sub.5RQQGERNREQGA

wherein: X1 is a D- or L-isomer of an amino acid selected from the group consisting of tyrosine ("Y"), or histidine ("H"); X.sub.2 is a D- or L-isomer of an amino acid selected from the group consisting of alanine ("A"), valine ("V"), or isoleucine ("I"); X.sub.3 is a D- or L-isomer of an amino acid selected from the group consisting of alanine ("A") or glycine ("G"); X.sub.4 is a D- or L-isomer of an amino acid selected from the group consisting of methionine ("M"), or leucine ("L"); X.sub.5 is a D- or L-isomer of an amino acid selected from the group consisting of serine ("S") or asparagines ("N"); and the isolated dGHRH is a biologically active polypeptide that has been engineered to contain a distinct amino acid sequence.

28. The composition of claim 27, wherein the isolated dGHRH increases growth hormone ("GH") in a recipient subject.

29. The composition of claim 27, wherein the isolated dGHRH improves hematological parameters in a recipient subject.

30. The composition of claim 27, wherein the isolated dGHRH thereof increases red blood cell values in a recipient subject.

31. The composition of claim 27, wherein the isolated dGHRH thereof increases hemoglobin values in a recipient subject.

32. The composition of claim 27, wherein the isolated dGHRH increases mean corpuscular hemoglobin values in a recipient subject.