MXPA04011766A

MXPA04011766A - Increased delivery of a nucleic acid constrtuct in vivo.

Info

Publication number: MXPA04011766A
Application number: MXPA04011766A
Authority: MX
Inventors: Hebel Henry
Original assignee: Advisys Inc
Priority date: 2002-05-28
Filing date: 2003-05-23
Publication date: 2005-03-31
Also published as: BR0311539A; CA2485976A1; CN1662261A; EP1513559A4; WO2003099341A1; AU2003273142A1; EP1513559A1

Abstract

Plasmid DNA delivered by injection / electroporation to the skeletal muscle can be expressed, and physiologic levels of transgene could be achieved into the circulation. Nevertheless, stabilization of naked DNA may be required and necessary in some cases, as prolonged storage at different temperatures before usage, injection into a large number of animals, etc. It is imperative that the associated compound should not be toxic to the cells (e.g. muscle cells) or cause breakage of plasmid DNA. It would be preferable for the coated DNA to have a similar or increased uptake into the target cells. Low molecular weight poly -L-glutamate compounds have all the desired properties. It was determined that mole/mole ratio DNA/PLG is the optimum concentration for gene therapeutic applications to the skeletal muscle, resulting in increased expression of the transgene, with no damage to the target tissue. Furthermore, stabilization of plasmid DNA by PLG has never been observed or described in the literature.

Description

INCREASED SUPPLY OF AN IN VIVO NUCLEIC ACID CONSTRUCTION BY THE POLI-L-G LUTAM ATO ("PLG") SYSTEM RELATED REQUESTS This application is a partial continuation of the US patent application serial number 10 / 156,670 entitled "PLASMID SUPPLEMENT BY MEANS OF GENE AND IN VIVO EXPRESSION OF THE POLI SYSTEM -LG LUTAM ATO (" PLG ")" and filed on 25 -5-2002 with Draghia-Akli and coinventores ^ registered as inventors, the entirety of whose application is incorporated to the present application specifically as a reference.

BACKGROUND OF THE INVENTION The supply of isolated or recombinant proteins has been used for many years to correct a formation of innate or acquired deficiencies and imbalances in subjects (for example, insulin in the case of diabetes). More recently, a nucleic acid expression construct having a specific encoded gene (ie, a plasmid) was delivered to a somatic tissue and has been shown to be useful for the correction of genetic deficiencies. Although both protein supplement methods work well, there are a number of advantages with the nucleic acid expression construction supplement method when compared to the administration of recombinant proteins, for example: the preservation of the native structure of the protein. protein, the improvement of biological activity, the avoidance of systemic toxicities, and the avoidance of infectious and toxic impurities. Additionally, the method of gene-mediated plasmid supplementation allows the subject to have a prolonged exposure to a therapeutic therapeutic-protein scale, as demonstrated by the persistent levels of the therapeutic protein found in the circulatory system of the subjects. The primary limitation of using recombinant protein is the restricted bioavailability of the recombinant protein after each administration. In contrast, the bioavailability of the gene-mediated plasmid supplement is not a problem because a single injection of plasmid in the skeletal muscle of the subject allows physiological expression for extended periods of time, as discussed in WO 99/05300 and WO. 01/06988. Plasmid DNA constructs are attractive candidates for direct supplementation therapy in the skeletal muscle of subjects because plasmid DNAs are well-defined entities, which are biochemically stable and have been used successfully for many years. The relatively low expression levels, achieved after a single injection of plasmid DNA, are often sufficient to test the bioactivity of the secreted peptides (Tsurumi and co-investigators, 1996). Although not expected to be bound by theory, injections of plasmid constructs can promote the production of enzymes and hormones in subjects in a way that mimics the natural process more clearly. Moreover, among the non-viral techniques for in vivo genetic product supplementation, the direct injection of plasmid DNA into the muscle tissue is simple, inexpensive, and safe. In contrast to viral vectors, a plasmid-based expression system can be composed of a synthetic gene delivery system, in addition to the nucleic acid encoding therapeutic gene products. In this way, many of the risks associated with viral vectors can be avoided. Plasmid products (ie, a non-viral expression system) 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 . To date, there have been no reported cases of plasmid vectors that have been integrated into a host chromosome (Ledwith and conivestigadores, 2000), which minimizes the risk of adverse effects such as the activation of oncogenes, or the inactivation of suppressor genes. tumors during the treatment. While episomal systems reside outside of chromosomes, plasmids have defined pharmacokinetic and elimination profiles, leading to a finite duration of gene expression in target tissues (Houk and coinvestigators, 2001).; Mahato and coinvestigadores, 1997). Unfortunately, most applications for plasmid supplementation by means of genes have suffered low levels of transgenic expression, which have resulted from the inefficient absorption of plasmid DNA in the cells of the treated tissues (Wells and co-investigators, 1997). Consequently, the use of plasmid DNA injected directly into a subject for therapy has been limited in the past. For example, the inefficient absorption of DNA in muscle fibers after simple direct injection has led to relatively low levels of expression, in normal, non-regenerative muscles (Vitadello and co-investigators, 1994) or ischemic (Takeshita and conivestigadores, 1996). Additionally, the duration of transgenic expression has been short (Hartikka and coinvestigadores, 1996) (Danko and Wolff, 1994). Until recently, the most successful previous clinical applications had been confined to vaccines (Davis and coinvestigators, 1994, Davis and coinvestigators, 1993). Thus, extensive efforts have been made during the past two decades to increase the supply of plasmid DNA to cells by both physical and chemical means (Danko and coinvestigators, 1994). For example, chemical means such as lipofectin / liposome fusion have been used; Polylysine condensation with and without adenovirus increase, with marginal success (Fixher and Wilson, 1994). The use of specific compositions consisting of polyacrylic acid has been discussed in the international patent publication WO 94/24983. The naked DNA has been administered as discussed in the international patent publication WO / 11092. Additionally, physical means of plasmid delivery include electrophoration, sonoporation and pressure. Although each of these methods has had limited success, of all the methods mentioned, electrophoration has been the most promising. Although one does not want to be bound by the theory, the supply of plasmid DNA in a cell by electrophoration involves the application of a pulsed voltage electric field to create transient pores in the cell membrane, which allow the influx of DNA molecules plasmid (Smith and Nordstrom, 2000). By adjusting the electrical pulse generated by an electrophoretic system, the efficiency of nucleic acid molecules traveling through passages or pores can be regulated. U.S. Patent 5,704,908 discloses an electrophoretic apparatus for delivering molecules to cells at a selected location within a cavity in the body of a patient. Pulsed voltage injection devices are also described in U.S. Patent Nos. 5,439,440 and 5,702,304, and PCT WO 96/12520, 96/12006, 95/19805, and 97/07826. The electrophoresis technique has previously been used to transfect tumor cells after the injection of plasmid DNA (Nishi and coinvestigators, 1997; Rols and conventors, 1998), or to deliver the anti-tumor drug bleomycin to cutaneous and subcutaneous tumors (Belehradek and coinvestigators, 1994, Heller and coinvestigadores, 1996). Electrophoration has also been used in rodents and other small animals, for example (Muramatsu and coinvestigators, 1998, Aihara and Miziyaki, 1998, Hasegawa and coinvestigators, 1998, Rizzuto and coinvestigators, 1999). Advanced techniques of intramuscular injections of plasmid DNA followed by electrophoresis in skeletal muscle have been shown to lead to high levels of circulation of growth hormone releasing hormone ("GHRH") (Draghia-Akli and coinvestigators, 1999) (Draghia- Akli and coinvestigadores, 2002b). The in vivo electrophoration of the skeletal muscle allows the plasmid DNA to be efficiently absorbed to induce the transient permeabilization of the pores of the bio-membrane, and allows the macromolecules, ions and water to pass from one side of the membrane to the other. In this way, electrophoration has been used to introduce drugs, DNA or other molecules into multicellular tissues. The technique has been used in vivo initially to transfect tumor cells after the injection of plasmid DNA (Rols and coinvestigators, 1998), or to deliver the anti-tumor drug bleomycin to cutaneous and subcutaneous tumors (Allegretti and Panje, 2001; Heller and coinvestigadores, 1996). Recently, numerous studies, mostly in small mammals, showed that the technique dramatically increases the uptake of plasmid by skeletal muscle cells, and allows the production of peptides at therapeutic levels (Yasui and coinvestigators, 2001; Yin and Tang, 2001). . Previously, we report that the human growth hormone releasing hormone ("GHRH") cDNA can be delivered to the skeletal muscle by injecting myogenic expression vector in mice and pigs, where it stimulates the secretion of human growth hormone (" GH ") for a period of at least two months (Draghia-Akli and co-investigators, 1997, Draghia-Akli and coinvestigators, 1999). Despite recent advances in plasmid DNA transfer technology, further improvements are needed in electrophoretic techniques and in plasmid DNA compositions. For example, in theory, the complete electrophoration procedure can be completed without causing permanent damage to the cell. However, in practice, the electrophoresis procedure inflicts a fatal strain on most cells and leads to the degradation of plasmid DNA (Hartikka and coinvestigators, 2001). Additionally, until now, the plasmids have been preserved at low temperature before use, due to the decrease in stability and degradation (Evans and coinvestigators, 2000). We have now optimized an electrophoresis delivery technique at a constant current and an Oplasmid DNA composition that prevents excessive cell damage and degradation of plasmid DNA during electrophoresis delivery in muscle cells. For example, during the electrophoretic process, a transfection facilitator polypeptide (e.g., poly-L-glutamate ("PLG")), increases the absorption process. Although you do not want to be bound by theory, you can use several mechanisms to increase absorption. For example, the transfection facilitator polypeptide may be bound to the surface of the proteins and facilitate absorption by increasing bioavailability, neutralizing the process of normal degradation in the interstitial fluid (e.g., protecting the DNA from the nucleases present in the interstitial fluid). In the cells, a transfection that facilitates the polypeptide can prevent the transport of DNA in the lysosomes (for example, organelles where the outer DNA and / or the proteins are degraded in the cells) by breaking the set of microtubules (Fujii and coinvestigators, 1986 ). Although one does not want to be bound by theory, transfection facilitator polypeptides (eg, PLG groups) naturally occur as side chain additions to proteins. Accordingly, polypeptides that facilitate transfection have been used to increase the stability of anticancer drugs (Li and coinvestigators, 2000), and as "glue" to close wounds or to prevent bleeding of tissues during repair. of wounds and tissues (Otani and coinvestigadores, 1998, Otani and coinvestigadores, 1996). Some transfection facilitator polypeptides (eg PLG) do not increase an immune response or the production of antibodies. It should be emphasized that some evidence suggests that certain transfection facilitator polypeptides can only be effective in conjunction with the electrophoresis method. Furthermore, PLGs have been shown to decrease the muscle damage associated with the supply of plasmids (Draghia-Akli and coinvestigators, 2002a). The efficient strategy of using transfection and electrophoration facilitating polypeptides to increase the electrophoretic delivery of a plasmid DNA construct has been described here and demonstrated in the skeletal muscle of three different mammalian species. The stability of the plasmid at high temperatures has been demonstrated.

BRIEF DESCRIPTION OF THE INVENTION One aspect of the present invention is a composition for facilitating the electrophoretic transfer of a nucleic acid expression construct in the cells of a receptor, wherein the nucleic acid construct can express a gene encoded in a receptor. The composition of the invention comprises a nucleic acid expression construct that is associated with a transfected transfection facilitator polypeptide. The composition is prepared such that the mole ratio of the transfection facilitator loaded polypeptide to the nucleic acid expression construct, comprises from 1 mole to 5,000 moles of the transfection facilitator loaded polypeptide per mole of nucleic acid expression construct. In a preferred embodiment, the ratio in molecules is equal to 1 mole of the nucleic acid expression construct to 1,200 moles or less of the transfection facilitator loaded polypeptide, and in another preferred embodiment, the mole ratio is equal to 1 mole of the construction of nucleic acid expression to 1 mole of the transfection facilitator loaded polypeptide. In a preferred embodiment, the transfection facilitator polypeptide comprises a charged polypeptide (eg, poly-L-glutamate). Additionally, the nucleic acid expression construct comprises SeqlD # 11, SeqlD # 12, SeqlD # 13, SeqlD # 14, SeqlD # 17, SeqlD # 18, SeqlD # 19, SeqlD # 20, SeqlD # 21. Additionally, the nucleic acid expression construct encodes a growth hormone releasing hormone ("GHRH") or a functional biological equivalent thereof, such as that included in HV-GHRH (SEQID # 1), TI-GHRH (SEQID # 2), TV-GHRH (SEQID # 3), 15/27/28-GHH (SEQID # 4), wt-GHRH (SEQID # 5). A second aspect of the present invention is a method for introducing a nucleic acid expression construct into a cell of a selected tissue in a receptor. The method comprises penetrating the selected tissue with a plurality of needle electrodes, wherein the plurality of needle electrodes are arranged in a spaced relationship; introducing a composition comprising nucleic acid expression construct and having a transfection facilitator loaded polypeptide, and applying an electrical pulse to the plurality of needle electrodes. However, calibration electrodes can also be used as an alternative for needle electrodes. The composition is prepared within a molar ratio of the transfection facilitator loaded polypeptide to nucleic acid expression construct comprising from 1 mole to 5,000 moles of the transfection facilitator loaded polypeptide per mole of the nucleic acid expression construct, up to 1,200 moles or less of the transfection facilitator loaded polypeptide, and in another preferred embodiment, the mole ratio is equal to 1 mole of the nucleic acid expression construct for 1 mole of the transfection facilitator loaded polypeptide. In a preferred embodiment, the transfection facilitator loaded polypeptide comprises a transfection facilitator loaded polypeptide (e.g., poly-L-glutamate). Additionally, the nucleic acid expression construct comprises SeqlD # 11, SeqlD # 12,, SeqlD # 13, SeqlD # 14, SeqlD # 17, SeqlD # 18, SeqlD # 19, SeqlD # 20, SeqlD # 21. Additionally, the nucleic acid expression construct encodes a growth hormone releasing hormone ("GHRH") or functional biological equivalent thereof, such as that included in HV-GHRH (SEQID # 1), TI-GHRH (SEQID # 2) ), TV-GHRH (SEQID # 3), 15/27/28-GH RH (SEQID # 4), wt-GHRH (SEQID # 5). A third aspect of the present invention is a method for increasing the stability of a nucleic acid expression construct, comprising: mixing the nucleic acid expression construct with a transfected transfection facilitator polypeptide, wherein the polypeptide loaded transfection facilitator 'comprises a poly-L-glutamate polypeptide and the expression construct of nucleic acid is used for gene supplementation by means of plasmid. The method involves making a composition that is prepared within a molar ratio of the transfection facilitator-loaded polypeptide to the nucleic acid expression construct comprising from 1 mol to 5 mol., 000 moles of the transfection facilitator loaded polypeptide per mole of nucleic acid expression construct, in a preferred embodiment, the mole ratio is equal to 1 mole of the nucleic acid expression construct up to 1,200 moles or less of the facilitator loaded polypeptide of transfection, and in another preferred embodiment, the mole ratio is equal to 1 mole of the nucleic acid expression construct to 1 mole of the transfected transfection facilitator polypeptide. In a preferred embodiment, the transfected transfection facilitator polypeptide comprises a transfected transfection facilitator polypeptide (eg, poly-L-glutamate). Additionally, the nucleic acid expression construct comprises SeqlD # 11, SeqlD # 12,, SeqlD # 13, SeqlD # 14, SeqlD # 17, SeqlD # 18, SeqlD # 19, SeqlD # 20, SeqlD # 21. Additionally, the nucleic acid expression construct encodes a growth hormone releasing hormone ("GHRH") or functional biological equivalent thereof, such as that included in HV-GHRH (SEQID # 1), TI-GHRH (SEQID # 2), TV-GHRH (SEQID # 3), 15/27/28-GH RH (SEQID # 4), wt-GHRH (SEQID # 5).

B REVE DESCRIPTION OF THE FIGURES Figure 1 shows an array of electrodes of earlier technology, which uses six electrodes in three opposite pairs. It also illustrates a single superimposed point of centralized electrophoration, which is the center point of the illustrated asterisk pattern. Figure 2 shows an array of electrodes of the present invention, using five electrodes. It further illustrates how a symmetric array of needle electrodes without opposing pairs can produce a decentralized pattern during an electrophoresis event in an area where congruent superimposed electrophorating points do not develop, and how an area of the decentralized pattern resembles a pentagon; Figure 3 shows serum levels of SEAP in mice that were injected with a pSP-SEAP expression plasmid coated with various concentrations of poly-L-glutamate; Figure 4 shows serum levels of SEAP in pigs that were injected with a pSP-SEAP expression plasmid covered with and without poly-L-glutamate. Figure 5 shows serum levels of SEAP in dogs that were injected with a pSP-SEAP expression plasmid covered with and without poly-L-glutamate. Figure 6 shows the in vitro stability of the increased plasmid DNA when poly-L-glutamate is added to the solution.

All samples were incubated for 6 months at 37 ° C.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES TERMS: The term "nucleic acid expression construct" as used herein, refers to any type of genetic construct comprising a nucleic acid encoded by an RNA capable of being transcribed. Alternatively, the term "expression vector" can be used. The term "functional biological equivalent" or GHRH as used herein, is a polypeptide that has been designed to contain a different amino acid sequence while simultaneously having similar or biologically enhanced activity when compared to the GHRH polypeptide. The term "encoded GHRH" as used herein, is a biologically active polypeptide. The term "delivery" as used herein, is defined as a means for introducing a material into a subject, a cell or any receptor, by means of chemical or biological processes, injection, mixing, electrophoration, sonoporation, or combination thereof. themselves, either under pressure or without it. The term "subject", as used here, refers to any species of the animal kingdom. In the preferred embodiments, it relates more specifically to humans and animals used as: pets (eg, cats, dogs, etc.); work (for example, horses, cows, etc.); food (chicken, fish, lambs, pigs, etc.); and all others known in the field. The term "receptor" as used herein, refers to any species of the animal kingdom. In preferred embodiments it refers more specifically to humans and animals used as: pets (eg, cats, dogs, etc.); work (for example, horses, cows, etc.); food (chicken, fish, lambs, pigs, etc.); and all others known in the field. The term "promoter" as used herein, refers to a DNA sequence that directs the transcription of a gene. A promoter can be "inducible", initiating transcription in response to an inducing agent, or, in contrast, a promoter can be "constitutive", characterized in that an inducing agent does not regulate the rate of transcription. A promoter can be regulated in a tissue-specific manner, or preferred for a tissue, so that it is active only to transcribe the operable linked coding region into a specific tissue type or types. 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 an amino acid sequence characteristic of a specific polypeptide. The term "analog" as used herein, includes any mutant of GHRH, or peptide fragments of GHRH present either synthetically or naturally, such as the forms HV-GHRH (SEQID # 1), Tl-GHRH (SEQID # 2), TV-GHRH (SEQID # 3), 15/27/28-GHRH (SEQID) # 4), (1-44) NH2 or (1-40) OH (SEQID # 6), or shorter forms up to (1-29) NH2. The term "growth hormone" ("GH"), as used herein, is defined as a hormone that refers to growth and acts as a chemical messenger to exert its action on a target cell. The term "growth hormone releasing hormone" ("GHRH") as used herein, is defined as a hormone that facilitates or stimulates the release of growth hormone, and to a lesser extent other pituitary hormones, such as prolactin. The term "molecular switch" as used herein, refers to a molecule released in a subject, which can regulate the transcription of a gene. The term "cassette", as used herein, is defined as one or more expression vectors. The term "post-injection", as used herein, refers to a period of time after the introduction of a nucleic acid cassette containing a heterologous nucleic acid sequence encoding GHRH or a biological equivalent thereof, in the cells of the subject and allows the expression of the encoded gene to occur while the modified cells are within the living organism.

The term "place" as used herein refers to the positioning of a plurality of electrodes (either plate or needle) in a selected tissue. The term "heterologous nucleic acid sequence" as used herein, is defined as a DNA sequence consisting of deferred regulation and expression elements. The term "vector" as used herein, refers to any vehicle that supplies nucleic acid in a cell or organism. Examples include plasmid vectors, viral vectors, liposomes, or cationic lipids. The term "electrophoration" as used herein, refers to a method that uses electrical pulses to deliver a nucleic acid sequence in cells. The term "electrical pulse" as used herein refers to a constant current pulse, or a constant voltage pulse. The term "poly-L-glutamate" ("PLG") as used herein, refers to a biodegradable polymer of L-glutamic acid, in some aspects of the present invention the sodium salt of said acid is suitable for use as a vector or adjuvant for the transfer of DNA in cells with or without electrophoresis. The term "spaced relation" as used herein, refers to positioning of electrodes in a tissue of a subject, either symmetrically or non-symmetrically with other electrodes. The term "weight ratio" as used herein, refers to an amount of nucleic acid expression construct (in micrograms), relative to an amount of polypeptide loaded transfection facilitator (in micrograms), in a composition, without take into account the total volume supplied. The term "molar ratio" as used herein, refers to an amount of nucleic acid expression (molar) construction relative to an amount of transfection facilitator-loaded polypeptide (in moles) in a composition. The standard one- and three-letter abbreviations for the amino acids used here are as follows: Alanine, A, ala; Arginine,, arg; Asparagine, N, asn; Aspartic acid, N, asp; Cysteine, C, cys; Glutamine, Q, gln; Glutamic acid, E, glu; Glycine, G, gli; Histidine, H, his; Isoleucine, I; Leucina, L, leu; Lysine, K, lys; Methionine, M, met; Phenylalanine, F, phe; Proline, P, pro; Serina, S, be; Threonine, T, thr; tryptophan, W, trp; Tyrosine, Y, Tyr; Valina, V, val. The ability of electrophoresis to increase the absorption of plasmid in skeletal muscle has been well documented. However, effective compositions of nucleic acid expression vectors and transfection facilitator agents for use in electrophoretic protocols have not been described in the literature. This invention characterizes the compositions and methods for increasing the delivery of a nucleic acid expression construct in a receptor.

FORMULATIONS OF COMPOSITION The ability of electrophoresis to improve the absorption of plasmids in skeletal muscle has been well documented, as described above. It has also been shown that other methods that do not involve electrophoresis improve the absorption of plasmids, for example, a plasmid formulated with poly-L-glutamate ("PLG") or polyvinylpyrrolidone (PVP ") particles facilitating transfection has been observed. increases genetic transfection and consequently increases gene expression up to 10-fold in muscles of mice, rats and dogs.An aspect of the present invention is the combination of electroforming and transfection facilitating particles associated with nucleic acid expression construction. that it is not desired to be bound by the theory, PLG will increase transfection of the plasmid during the electrophoration process, not only by physical stabilization of the plasmid DNA, and facilitation of intracellular transport through the pores of the membrane, but also to through an active transport mechanism, for example, the surface proteins loaded ositivamente in the cells, attract and compose negatively charged PLG bound to plasmid DNA through protein-protein interactions. When an electric field is applied, the surface proteins reverse the direction and actively internalize the DNA molecules. Additionally, the PLG / DNA molecules that are in contact with the surface of the cell, only need to migrate through the plasma membrane, while the opposite to the DNA molecules are located outside the cell surface in the intracellular space. Thus, protein-protein interactions and the proximity of transfection particles can substantially increase transfection efficiency. Poly-L-g lutamate ("PLG") is a stable compound, and resistant to high temperatures, denaturing. PLG has previously been used to increase stability in vaccine preparations because it does not increase the immunogenicity of vaccines. Additionally, PLG has been used as an antitoxin by post-antigen inhalation or exposure to high amounts of ozone. Plasmid DNAs supplied by injection, electrophoresis or both in skeletal muscle are easily expressed, and can be measured as indicated by the physiological levels of the transgenic product in the circulation. However, naked DNA stabilization may be required and is necessary in some cases, such as prolonged storage before use, injection into a large number of animals. As the plasmid DNA can be stored at different temperatures for varying periods of time, it is critical that the plasmid solutions be stable for extended periods of time. It is important that the compound associated with the DNA is not toxic to the cells (for example muscle cells), and does not cause the breaking of the plasmid DNA. It would be preferable for the plasmid DNA composition and the associated transfection facilitator particles to have a similar or increased absorption in the target cells. This invention uses low concentrations (eg, below 6 μm / μ ?, preferably about 0.01 μ / μm) of low and medium molecular weight poly-L-glutamate compounds (eg 1-15 kDa) , with an average of 10 kDa or 15-50 kDa, with an average of 35 kDa) showing all the desired properties for an effective composition of nucleic acid expression vector and transfection facilitator polypeptide. Although PLG can be used at a high concentration in non-electrophoretic applications. We have determined that the low molar ratio of nucleic acid expression vector to PLG is optimal for electrophoretic applications for skeletal muscle. An example of a molar ratio of nucleic acid expression vector useful with respect to PLG is about 1: 5,000. Another example of a more useful proportion of nucleic acid expression vector relative to PLG comprises about 1: 2,500. An example of a preferred molar ratio of nucleic acid expression vector to PLG is about 1: 1,200. An illustrative molar ratio of nucleic acid expression vector to PLG comprises one of about 1: 800. An illustrative molar ratio of nucleic acid expression vector to PLG comprises one of about 1: 500. An example of a selected molar ratio of nucleic acid expression vector to PLG comprises one of about 1: 200. Another example of an even more selected molar ratio of nucleic acid expression vector to PLG comprises one of approximately 1: 100 An example of a preference molar ratio! of nucleic acid expression vector with respect to PLG comprises one of about 1:50. Another example of a more preferential molar ratio of nucleic acid expression vector to PLG comprises one of about 1:20. An example of an even more preferential molar ratio of nucleic acid expression vector with respect to PLG comprises one of about 1: 10- An example of a more preferred molar ratio of nucleic acid expression vector with respect to PLG is approximately 1: 1. The appropriate molar ratio can be calculated by the moles of a nucleic acid expression vector of appropriate average length (for example in the scale of 2)., 000 bp to 30,000 bp) with respect to the PLG moles of low and medium molecular weight poly-L-glutamate (for example 1-15 kDa, with an average of 10 kDa or 15-50 kDa, with an average of 35 kDa). The resulting electrophoration of a plasmid DNA associated with the PLG composition resulted in an increased expression of a reporter transgene and no damage to the target tissue. Accordingly, the pharmaceutical composition of the The present invention can be delivered by several routes and to several sites in an animal body to achieve a particular effect. One skilled in the art will recognize that although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. Although not wishing to be bound by theory, local or systemic delivery can be achieved by administration comprising application or instillation of the composition formulated in the body cavities, inhalation or insufflation by an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous intradermal, as well as topical administration. Additionally, different delivery methods can be used to administer a plasmid facilitating agent composition in a cell. Examples include: (1) methods that use physical means, such as electrophoration (electricity), a gene gun (physical force), or application of large volumes of a liquid (pressure); and (2) methods wherein said vector is composed with another entity, such as a liposome or carrier molecule.

CONSTANT CURRENT ELECTROFORATION It is believed that the base phenomenon of electrophoration is the same in all cases, but the exact mechanism responsible for the observed effects has not been elucidated. Although you do not want to be bound by theory, the open manifestation of the electrophoretic effect is that cell membranes become transiently permeable to large molecules, after the cells have been exposed to electrical pulses. There are conduits through the walls, which under normal circumstances maintain a transmembrane potential at rest of ca. 90 mV allowing bidirectional ion migration. Although you do not want to be bound by the theory, electrophoresis makes use of the same structures, forcing a high ionic flow through these structures and opening or lengthening the ducts. In previous technologies, metallic electrodes are placed in contact with the tissues and are supplied with predetermined voltages, proportional to the distance between the electrodes. The protocols used for electrophoration are defined in terms of the resulting field strengths, according to the formula E = V / d, where ("E") is the field, ("V") is the imposed voltage and ("d") is the distance between the electrodes. The intensity of the electric field E has been a very important value in previous techniques when formulating electrophoresis protocols for the delivery of a drug or macromolecule in the subject's cell. Accordingly, it is possible to calculate any electric field strength for a variety of protocols by applying a predetermined voltage pulse that is proportional to the distance between the electrodes. However, one caveat is that an electric field can be generated in a tissue with isolated electrodes (ie, the flow of ions is not necessary to create an electric field). Although you do not want to be bound by theory, it is the current that is needed for successful electrophoresis, not the electric field per se.

During electrophoration, the heat produced is the product of the impedance between electrodes, the square of the current, and the duration of the pulse. Heat is produced during electrophoresis in tissues, and can be derived as the product of current between electrodes, voltage and pulse duration. The protocols currently described for electrophoration are defined in terms of the resulting field strengths E, which are dependent on short voltage pulses of unknown current. Accordingly, the resistance or heat generated in a tissue can not be determined, which leads to various successes with different pulsed voltage electrophoration protocols with predetermined voltages. The ability to mimic the heat of the cells through the electrodes can increase the effectiveness of any voltage pulse protocol for electrophoresis. The control of the current flow between electrodes allows to determine the relative heating of the cells. A) Yes, it is the current that determines the subsequent effectiveness of any given pulse protocol, and not the voltage across the electrodes. The predetermined voltages do not produce predetermined currents, and the prior art does not provide a means to determine the exact current dose, which limits the utility of the technique. Thus, controlling and maintaining the current in the tissue between two electrodes below a threshold will allow varying pulse conditions, reduce cell heating, create less cell death, and incorporate macromolecules into cells more efficiently when compared to predetermined voltage pulses. . A device for constant current electrophoresis is the invention of a pending patent application entitled "Set of electrodes for constant current electrophoresis and their use" S / N 60 / 362,362 received on March 7, 2002 with Westerstein and co-inventors (the "Western application 362") listed as inventors, and is incorporated herein by reference. One aspect of the Western application 362 solves the above problem by providing a means to effectively control the dose of electricity delivered to the cells in the inter-electrode space by the precise control of the ion flux that strikes the conduits in the cell membranes. Thus, the precise dose of electricity to the tissues can be calculated as the product of the current level, the length of the pulse and the number of pulses supplied. The constant current system comprises an electrode apparatus connected to a specially designed circuit, which is also used in the present invention. One aspect of the present invention is to provide a means for delivering the electrophoretic current to a tissue volume along a plurality of pathways without causing excessive concentration of accumulated current at any other location, thereby preventing cell death due to overheating. of the tissue. However, the composition of the nucleic acid expression vector associated with a transfection facilitating polypeptide will further facilitate successful transfection protocols. For example, the maximum energy supply from a particular pulse could occur along a line connecting two electrodes. An example of the energy supply path in a prior art electrode, which uses three pairs of radial electrodes with a central electrode, is described above as shown in Figure 1. A distribution of energy crosses the center point of the electrodes. electrodes, which can lead to unnecessary heating and decreased cell survival. Thus, the nucleic acid transfection facilitation composition of the present invention can also help to stabilize the cells in electrophoretic protocols of previous technologies. The electrodes of one embodiment of the present invention are arranged in a radial and symmetric array, but unless they are from earlier technologies, the electrodes are numbered oddly, and not in opposite pairs (Figure 2). The supply of an electrical pulse to any two of the electrodes of an electric pulse generator results in a pattern that is best described as a polygon. Tracing this pattern could result in a five-pointed star with a pentagon of electrical pulses surrounding the center of the array in the tissue where the concentration of the molecules to be transfected is greater. Although you do not want to be bound by the theory, it is not the odd number of electrodes, per se, that makes a difference, but the direction of the current paths. With the configuration of previous techniques, all pulses generate a current that passes through the center of the assembly. The accumulated dose, that is, the heating effect, is concentrated in the center, with the peripheral dose falling rapidly. With the arrangement of "five-point star" the dose is spread more evenly, on a larger volume. For example, if the electrodes are arranged in an array of five electrodes, the pulses could be applied sequentially to electrodes 1 and 3, then 3 and 5, then 5 and 2, then 2 and 4, then 4 and 1. However , because the tissue between the electrodes is a volume conductor, certain intensity of current exists along the parallel lines, weakening as the distance from the centerline increases. The cumulative effect of a pulse sequence results in a more even distribution of the energy delivered to the tissues, increasing the probability that the cells that have been electrophoresed actually survive the procedure. In prior art it is known that the nature of the voltage pulse to be generated is determined by the nature of the tissue, the size of the selected tissue and the distance between the electrodes. It is desirable that the voltage pulse be as homologous as possible and of the correct amplitude. The excessive strength of the field results in the lysis of the cells, while a low field strength results in a reduced efficiency of electrophoration. Inventions of previous techniques use the distance between electrodes to calculate the electric field strength and the predetermined voltage pulses for electrophoration. This confidence in knowing the distance between the electrodes is a limitation for the design of electrodes. Because the programmable current pulse controller will determine the impedance in a tissue volume between two electrodes, the distance between electrodes is not a critical factor in determining the appropriate electric current pulse. Accordingly, an alternative embodiment of the needle electrode array design would be one that is not symmetric. Additionally, a person skilled in the art can imagine any number of symmetrical and non-symmetrical needle electrode arrays that do not deviate from the spirit and scope of a particular electrode design. The depth of each individual electrode within an array and in the desired tissue can be varied with comparable results. Additionally, multiple injection sites for the macromolecules can be added to the needle electrode array. Using the constant current electrophoresis device described in Western application 362, a simple means is available to determine the temperature rise of the tissues exposed to the pulses. For example, the product of the impedance measured between electrodes, the square of the current and the duration of the accumulated pulse is a measure of the total energy supplied. This amount can be converted to Celsius degrees when the volume of the tissues encompassed by the electrodes and the specific heat of the tissues is known. For example, the increase in tissue temperature ("T", Celsius) is the resistance ("B", ohms), current ("I", amps), pulse length ("t", seconds), and the conversion factor between joules and calories (K ") T = RI2tK At the moment of electrophoration, the current increases in a previous technology system, where a predetermined voltage has been imposed on the electrodes, due to the fact that the permeability Increased cellular low impedance between electrodes.This can lead to an excessive temperature increase, resulting in cell death.For example, using common values for conventional electrophores, and assuming that the volume covered by the electrodes is one cubic centimeter and the Tissue-specific heat is close to the unit, the increase in temperature due to a 50 msec pulse with an average current of 5 Amps through a typical load impedance of 25 ohms is ca 7.5 ° C. This points to the need to insert an adequate delay between successive pulses, to allow the circulatory system of the subjects to eliminate enough heat, so that the accumulated temperature increase does not result in the destruction of the tissues that are being electrophoresed. The advantage of a constant current is that the pulse can be prevented from reaching an amplitude at which the cells are destroyed. In a predetermined voltage system, the current can reach a destructive intensity, and the operator can not prevent this from happening. In a constant current system, the current is preset below a level threshold where cell death does not occur. The exact setting of the current depends on the configuration of the electrode, and must be determined experimentally. However, once the appropriate level has been determined, cell survival is ensured, if necessary. The addition of a nucleic acid expression construct associated with a transfection facilitating polypeptide enhances the opportunity of the electrophored cells to incorporate the plasmid construct. Nucleic acid constructs for therapy: One aspect of this invention relates to a composition and method for the efficient delivery of a nucleic acid construct for a tissue as a treatment for various diseases found in chronically ill subjects. More specifically, the aspects of this invention pertain to a method for delivering a heterologous nucleic acid sequence encoding a specific gene (eg, growth hormone releasing hormone ("GHRH") or its biological equivalent) in one or more cells of the subject (eg, somatic, stem or germ cells) and allows the expression of the encoded gene (eg, GHRH or its biological equivalent) to occur while the modified cells are within the subject. The method of supplying the nucleic acid sequence encoding the gene is by electrophoration. Subsequent expression of the encoded gene can be regulated by a specific tissue promoter (eg, muscle), and / or by a regulatory protein that contains a modified ligand binding domain (eg, mifepistone), is administered externally in the subject . For example, the expression extracranial release resulting from GHRH or its biological equivalent by the modified cells can be used to treat anemia, exhaustion, immune dysfunction, extension of life and other disorders in the chronically ill subject. Recombinant GH replacement therapy is widely used clinically, with beneficial effects, but generally, doses with supraphysiological. These high doses of recombinant GH are associated with deleterious side effects, for example, up to 30% of patients treated with recombinant GH report a high frequency of insulin resistance or accelerated growth and closure of bone epiphyses in pediatric patients. Additionally, the molecular heterogeneity of circulating GH has important implications for growth and homeostasis, which can lead to a less potent GH that has a reduced ability to stimulate the prolactin receptor. These unwanted side effects result from the fact that the treatment with recombinant exogenous GH protein increases the basal levels of GH and eliminates the natural episodic GH pulses. On the contrary, no side effects have been reported for recombinant GHRH therapies. Normal levels of GHRH in the circulation of the pituitary portal reach from 150 to 180 pg / ml, while the values of the hormone in the circulatory system are from 100 to 500 pg / ml. Some patients with acromegaly caused by extracranial tumors have a level that is about 100 times as high (for example 50 ng / ml of GHRH immunoreactive). Long-term studies using recombinant GHRH therapies (1 to 5 years) in children and the elderly have shown an absence of the classic side effects of CH, such as changes in fasting glucose concentration, or, in pediatric patients, the accelerated growth of the epiphyseal bone and the closing or sliding of the essential femoral epiphysis. Thus, recombinant GHRH therapy may be more physiological than recombinant GH therapy. Unfortunately, due to the short half-life of the peptide in vivo, frequent intravenous or subcutaneous administration (ie, one to three times a day) is needed if the recombinant protein is used. A method of genetic transfer, however, could overcome these limitations to the use of GHRH. Moreover, a broad dose scale can be therapeutic. The choice of GHRH for a genetic therapeutic application is favored by the fact that the gene, cDNA and several mutated and native molecules have been characterized for pig and other species, and the measurement of therapeutic efficacy is direct and unambiguous. 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 Plasmid vectors containing the specific SPc5-12 muscle-specific promoter were previously described (Li and coauthors, 1999). The wild-type and mutated porcine GHRH cDNAs were generated by site-directed mutagenesis of the site-directed GHRH cDNA (Altered site mutagenesis system of altered sites II, Promega, Madison, Wl), and cloned into the BamHI / Hind III sites of pSPc5 -12, to generate pSP-wt-GHRH, or pSP-HV-GHRH respectively. The untranslated 3 '(3'UTR) region of the growth hormone was cloned downstream of the GHRH cDNA. The resulting plasmids contained the mutant coding region for GHRH, and the amino acid sequences were not naturally present in mammals. Although you do not want to be bound by the theory, the effects in the treatment of anemia; total increase in red blood cell maca in a subject; reverse the exhaustion; Reverse the loss of abnormal weight; treat immune dysfunction; reverse the suppression of lymphopoiesis; or extend the life expectancy for the chronically ill subject, being ultimately determined by the levels of circulation of analogous GHRH hormones. Several different plasmids encoding different amino acid sequences of mutated GHRH or their biological equivalents are the following: Plasmid Amino acid encoded sequence wt-GHRH YADAIFTNSYRKVLGQLSARKLLQDIMSROOGERNOFOGA-OH (SEQID # 5) HV-GHRH HVDAIFTNS YRKVLAQLSARKLLQD ILNRQQGERNQEQG A- OH (SEQID # 1) IT -GHRH YIDAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQGA-OH (SEQID # 2) TV-GHRH YVDAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQGA-OH (SEQID # 3) 15/57 / 28- YADAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQGA-GHRH OH (SEQID # 4) In general, the encoded GHRH or its functional biological equivalent is of the formula (SeqlD # 6): -A.1-A2-DAIFTNSYRKVL-A3-QLSARKLLQDI-A4-A5-RQQGERNQEQGA-OH wherein: a one-letter abbreviation standard is used, and A, is a D- or L- isomer of an amino acid selected from the group consisting of tyrosine ("Y"), or histidine ("H"); A2 is an L- or D-isomer of an amino acid selected from the group consisting of alanine ("A"), valine ("V") or isoleucine ("I"); A3 is an O- or L- isomer of an amino acid selected from the group consisting of alanine ("A") or glycine ("G"); A4 is a D- or L- Isomer of an amino acid selected from the group consisting of methiotein ("M"), or leucine ("L"); A5 is a D- or L- isomer of an amino acid selected from the group consisting of serine ("S") or asparagine ("N"). Another plasmid that was used included the pSP-SEAP construct containing the Sacl / HindIII SPc5-12 fragment, SEAP gene and SV40 3'UTR of the pSEAP-2 basic vector (Clontech Laboratories, Inc., Palo Alto, CA). The plasmids described above do not contain a polylinker, an IGF-I gene, a 7NCR (unencoded region) skeletal promoter -actin3'UTR (untranslated region). Additionally, these plasmids were introduced by muscle injection, followed by electrophoresis in vivo, as described below. In terms of "biological functional equivalents" those skilled in the art well understand that, in the definition of a protein and / or polynucleotide "biological functional equivalent", it is inherent that there is a limit on the number of changes that can be made in a portion of the molecule while retaining a molecule with an acceptable equivalent biological activity level. The functional biological equivalents accordingly are defined herein as those proteins (and polynucleotides) in selected amino acids (or codons) that can be substituted. A peptide comprising a functional biological equivalent of GHRH is a polypeptide that has been designed to contain different amino acid sequences while simultaneously having similar or improved biological activity when compared to GHRH. For example, a biological activity of GHRH is to facilitate the secretion of growth hormone ("GH") in the subject.

PLASMID ASSOCIATED WITH PLG IN MICE In order to demonstrate improved absorption of the electrophored cells with a composition of a nucleic acid expression construct associated with a transfection facilitating polypeptide, a series of electrophoretic experiments were designed. Three sets of separate experiments were performed in mice. All mice were given a total of 30 μg (micrograms) of pSP-SEAP (approximately 5,000 base pairs ("bp")), +/- PLG (mean heavy PM 10,900) in n total volume of 25 μL · ( mieroliters). A group of 10 mice received naked plasmid, without cover; the following groups received covered plasmid with decreasing concentrations of PLG (See Table 1, below): The molar proportions are provided for purposes of the example. The molar proportions indicated in Table 1 are based on a 5,000 bp nucleic acid expression vector, and the PLGs with the heavy average molecular weight of 10,900. For example, group 2 in Table 1 has a total injection of 30 μg of DNA vector associated with 0.25 μg of transfection facilitator polypeptides, wherein the molar ratio is less than 1: 2. Molar proportions of the vector DNA to PLG having a 1: 1 ratio comprise a lower bound formulation that still have a higher transfection efficiency than a naked DNA vector alone. A person skilled in the art is able to formulate molar ratio calculations with different lengths of expression vectors and variable molecular weights of PLG. Additionally, it is to be understood that the length of the nucleic acid expression vector and the heavy average molecular weight of the PLGs are subject to change based on specific vector lengths and particular formulation strategies known to one skilled in the art (e.g. functional nucleic acid expression vectors greater than or less than about 5,000 nucleotides, and PLGs with an average molecular weight less than about 1 I to about 30 kDa). Accordingly, even the smallest PLG polymers (eg, the trimers having a molecular weight ~ 400 Da) can be used for this invention. Electrophoration was carried out using a constant current electrophoretic apparatus which is the subject of the pending patent application Western 362. This device was used to deliver square wave pulses in all the experiments. Amplitude conditions of 1mA, 5 pulses, 50 milliseconds per pulse were used. Gauge electrodes were used to deliver the electrical pulses live. The gauge electrodes (plate) consisted of 1.5 cm square metal blocks mounted on a ruler, so that the distance between the plates could be easily evaluated. Plasmid DNA or associated DNA was injected through the intact skin into the mouse tibialis anterior muscle. Each animal received an injection at a single injection site. Although a constant current electroforming device was used in specific examples, it is not intended to limit the general embodiments of the invention (ie, other electrophoresis devices can provide satisfactory results). Moreover, the order of placement of the electrodes and the subsequent injection of the plasmid are not sequentially limiting. In order to determine the expression amount of the SEAP gene that was encoded in the DNA vector, the mice were bled and the serum was collected for up to 3 months post-injection. The SEAP molecule usually disappears after birth, and is immunogenic in adult animals. The blood was collected by tail vein collection for mice, before plasmid administration, and up to 3 months post-injection in the mice. SEAP serum levels were determined using a chemiluminescence assay (Tropix, Bedford, MA), following the manufacturer's instructions. Figure 3 shows serum levels of SEAP for all five groups of mice described in Table 1. Even though the naked plasmid (Group 1), Figure 3) showed some expression, all groups with the nucleic acid expression vector associated with PLG (groups 2-5, Figure 3), showed significantly higher serum SEAP levels. However, when samples from animals selected from each group were analyzed with histochemistry for markers of inflammation (e.g., macrophages, B cells and counterstained with hematoxylin / eosin), mice of group 5, (ie, acid expression construct) nucleic acid coated with 0.01 μg μL of PLG) had the lowest inflammation associated with the 3-day post-injection delivery procedure. Despite the highest expression at early time points, group 2 injected with plasmid associated with 6μg / μL had high inflammation and some morphological changes. This observation corresponds to the data in the literature, which show extended expression in the short term using PLG compounds, expression that disappears in approximately 1 month post-injection. (Fewell and coinvestigadores, 2001).

HISTOLOGICAL ANALYSIS Dehydrated muscle and skin samples were placed in alcohol overnight and immersed in paraffin. Sections of five microns were cut and stained with hematoxylin / eosin (Sigma Chemical, St. Louis, MO). The serial sections were stained with picric acid. Digital images of the slides were captured using a CoolSnap color digital camera (Roper Scientific, Tucson, AZ) with MetaMorph software (Universal Imaging Corporation, Downington, PA) and a Zeiss Axioplan 2 microscope with one objective (x40) (numerical aperture 0.75). plan).

STATISTICS The data was analyzed using the STATISTICA analysis package (StatSoft, Inc. Tulsa, OK). The values shown in the figures are the mean ± s.e.m. Specific values of P were obtained by comparison using ANOVA. A P < 0.05 as a level of statistical significance.

EXAMPLE 2: PLG COVER IN PIGS In order to demonstrate similar results in a larger mammal, experiments similar to Example 1 were carried out in pigs. Thus, two groups of three pigs were injected with 500 μg (micrograms) of pSP-SEAP and electrophoresed. The plasmid expressed secreted embryonic alkaline phosphatase ("SEAP"). The molecule usually disappears after birth, and is immunogenic in adult animals. One group received naked nucleic acid expression construct and the second group received nucleic acid construction at 0.01 μ9 / μg - PLG. The pigs were weighed and bled before the injection, and each of the other days until day 10 after injection. Serum was collected from the pigs by jugular puncture before the plasmid injection, and at 2, 4, 6, 8 and 10 days for the SEAP studies. Serum serum levels were determined using a chemiluminescence assay (Tropix, Bedford, MA) following the manufacturer's instructions. The SEAP assay (Figure 4) showed an increased expression in the animals injected with plasmid coated PLG against the naked plasmid during the 12 days of the experiment (32.9 ± 19.3 ng / mL / kg in the pigs of the plasmid / PLG against 17.14 ± 12.44 ng / mL / kg in animals injected with naked plasmid). Although one does not want to be bound by the theory, the increased expression can be attributed to the increased stability of the plasmid, facilitation of transfection in the muscle cells, or both.

ELECTROFORATION DEVICES A constant-current electroforming machine (Advisys, Inc.) was used to supply square-wave pulses in all experiments. The electrophoration parameters included an amplitude condition of 1 mA, 5 pulses, 50 milliseconds per pulse. A needle electrode was used to deliver the electrical pulses in vivo. The 5-needle electrode device consists of a circular arrangement (1 cm in diameter) of equally spaced 21-gauge needles mounted on a non-conductive material. All needles were two centimeters long and during all injections or electrophoresis, the needles were inserted completely into the muscle. Plasmid DNA was injected through the intact skin into the semi-tendinous muscle of pigs with a 21-gauge needle. Each animal received an injection at a single injection site and the injection site also received a tattoo to be isolated easily at the end of the experiment.

HISTOLOGICAL ANALYSIS Muscle and skin samples were fixed overnight, dehydrated in alcohol and immersed in paraffin. Sections of five microns were cut and stained with hematoxylin / eosin (Sigma). The serial sections were stained with picric acid. Digital images of the slides were captured using a CoolSnap color digital camera (Roper Scientific, Tucson, AZ) with Meta Morph software (Universal Imaging Corporation, Downington, PA) and a Zeiss Axioplan 2 microscope with a (x40) objective (numerical aperture). 0.75 plan).

EXAMPLE 3: PLG COVER IN DOGS In order to demonstrate similar results in different species of larger mammals, experiments similar to Example 2 were conducted in dogs. Thus, a comparison of expression in dogs injected with electrodes in a 5-needle array, with plasmid covered or naked. Four groups of five dogs were injected with a plasmid DNA, pSP-SEAP, expressing the secreted embryonic alkaline phosphatase ("SEAP"). The molecule usually disappears after birth, and is immunogenic in adult animals. No adverse reaction, or change in biochemical, clinical and hormonal profiles, is related to the development of the immune response to SEAP in animals. As described above, the injection was followed by electrophoresis, using standard conditions and five-needle electrodes. The plasmid DNA was naked or covered, with a mole / mole dilution of poly-L-glutamate. The groups are as follows: Group 1 - 5 needles (5N), 0.5 mg, nude (NK) Group 2 - 5 needles (5N), 0.1 mg, nude (NK) Group 3 - 5 needles (5N), 0.5 mg, covered (PLG) Group 4 - 5 needles (5N), 0.1 mg, covered (PLG) Dogs were weighed and bled at the start (pre-injection) and all other days until day 10 post-injection. SEAP tests were performed on the serum. The values were corrected by weight (blood volume). The differences in SEAP values between the different groups injected were analyzed. The results of this experiment are shown in Figure 5. The results showed that a needle electrode can be used in dogs to efficiently mediate electrophoresis. Additionally, the DNA coated with PLG increases the stability of the plasmid and the electrophoration efficiency in dogs.

EXAMPLE 4: PLG INCREASES THE STABILITY OF IN VITRO PLASID AT HIGH TEMPERATURES In order to evaluate the effects of PLG on the stability of the plasmid, the following tests were performed. It was diluted in distilled water, pSP-HV-GHRH plasmid encoded by a supra porcine growth hormone release hormone, at a final concentration of 2 mg / ml. PLG was added in a mol / mol ratio to a group of samples, while pLG was not added to the control samples. All samples were incubated for 6 months at 37 ° C. After 6 months, aliquots of all samples were taken, and spread on agarose gel (Figure 6). as seen in the gel image, all the plasmid is present in the samples to which PLCs were added, whereas in the control samples all the plasmid is completely degraded. A person skilled in the art will readily appreciate that the patent of the invention is well adapted to achieve the objects and obtain the ends and advantages also mentioned as inherent in the present. Growth hormone, growth hormone releasing hormone, analogs, plasmids, vectors, transfection facilitator-loaded polypeptides, poly-L-glutamate, pharmaceutical compositions, treatments, electrophoretic methods, procedures and other techniques described herein are currently representative of various aspects of the present invention and are intended to serve as an example and not as scope limitations. Changes in the present and other uses will occur to those skilled in the art, which are comprised within the spirit of the invention or defined by the scope of the pending claims. Accordingly, the present invention provides a method for transferring a therapeutic gene to a host, which comprises administering the vector of the present invention, preferably as part of a composition, using any of the aforementioned routes of administration known to the experts in the field, and appropriate for a particular application. The effective genetic transfer of a vector to a host cell according to the present invention can be monitored in terms of a therapeutic effect (e.g. relief of some symptom associated with the particular disease being treated) or, additionally, by evidence of the transferred gene or gene expression in the host (eg, using the polymerase chain reaction in conjunction with sequencing, North or South hybridizations, or transcription assays to detect the nucleic acid in the host cells, or use immunological analyzes with inkblot, detection by means of antibodies, studies of mRNA or half-life protein, or assays particularized to detect the protein or polypeptide encoded by the transferred nucleic acid, or imbedded in the level or function due to this transfer). These methods described herein in no way include all, and additional methods for the specific application will be apparent to those skilled in the art. Moreover, the effective amount of the compositions can be further approximated by analogy to known compounds to achieve the desired effect. Additionally, the actual dose and schedule may vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depend on differences between individuals in pharmacokinetics, drug disposition, and metabolism. Similarly, amounts may vary in in vitro applications depending on the particular cell line used (e.g., based on the number of vector receptors present on the cell surface, or the ability of the particular vector employed by the transfer In addition, the amount of vector to be added per cell will probably vary with the length and stability of the therapeutic gene inserted in the vector, as well as the nature of the sequence, and particularly it is a parameter that it needs to be determined empirically, and it can be altered due to factors not inherent in the methods of the present invention (for example, the cost associated with the synthesis.) A person skilled in the art can easily make any necessary adjustments according to the requirements of the particular situation.

REFERENCES CITED The following documents and US patent publications are incorporated herein by reference.

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(1996) Increased expression of direct gene transfer skeletal muscles observed in the acute ischemic injury in rats, Laboratory Investigation 74, 1061-1065 Tsurumi, Y. , Takeshita, S., Chen, D., Kearney,., Rossow, ST, Passeri, J., Horowitz, JR, Symes, JF, and Isner, JM (1996) Direct intramuscular gene transfer of naked DNA encoding bascular endothelial growth factor augments collateral development and tissue perfusion [see comments] Circulation 94, 3281-3290, Vitadello.M., Schiaffino, MV, rd, A., Scarpa, M., and Schiafinno, S. (1994). Gene transfer regenerating muscle. Human Gene Therapy, 5, 11-18. Wells, K.E., Maule, J., Kingston, R., Foster, K.

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Claims

CLAIMS 1. A composition comprising: (a) a nucleic acid expression construct; and (b) a loaded transfection facilitator polypeptide thereto; characterized in that a molar ratio of the transfection facilitator loaded polypeptide to the nucleic acid expression construct comprises from 1 mole to 5,000 moles of the transfection facilitator loaded polypeptide per mole of nucleic acid expression construct. The composition of claim 1, further characterized in that the transfection facilitator loaded polypeptide comprises poly-L-glutamate. The composition of claim 1, further characterized in that the mole ratio of the transfection facilitator loaded polypeptide to the nucleic acid expression construct is equal to 1,200 moles or less of the transfection facilitator loaded polypeptide per mole of the construction of nucleic acid expression. 4. The composition of claim 1, further characterized in that the mole ratio of the transfection facilitator loaded polypeptide to the nucleic acid expression construct is equal to 1 mole of the transfection facilitator loaded polypeptide per mole of expression construct. nucleic acid. The composition of claim 1, further characterized in that an average molecular length of the nucleic acid expression vector is from about 2,000 to about 5,000 base pairs nucleotides. The composition of claim 1, further characterized in that an average molecular weight of the transfection facilitating transcription polypeptide is from about 400 to about 30,000 Da. The composition of claim 1, further characterized in that an average molecular length of the nucleic acid expression vector is about 5,000 nucleotide base pairs, and an average molecular weight of the transfection facilitating transcription polypeptide is approximately 10,900 Da. The composition of claim 1, further characterized in that the nucleic acid expression construct comprises SeqlD # 11, SeqlD # 12, SeqlD # 13, SeqlD # 14, SeqlD # 17, SeqlD # 18, SeqlD # 19, SeqlD # 20, SeqlD # 21. The composition of claim 1, further characterized in that the nucleic acid expression construct comprises a gene encoding a growth hormone releasing hormone ("GHRH") or its biological functional equivalent. The composition of claim 9, further characterized in that the encoded GHRH is a biologically active polypeptide, and the functional biological encoded equivalent of GHRH is a polypeptide that has been designed to contain a distinct amino acid sequence while simultaneously having similar or enhanced biological activity when compared to the GHRH polypeptide. 11. The composition of claim 9, further characterized in that the biological functional equivalent of the encoded GHRH is of the formula (SEQID # 6): -X-1-X2-DAIFTNSYRKVL-X3-QLSARKLLQDI-X4-X5-RQQGERNQEQGA-OH wherein the formula has the following characteristics: Xi is a D- or L- isomer of the amino acid tyrosine ("Y") or histidine ("H"); X2 is a D- or L- isomer of the amino acid alanine ("A"), valine ("V") or isoleucine ("I"); X3 is a D- or L- isomer of the amino acid alanine ("A") or glycine ("G"); X4 is a D- or L- isomer of the amino acid methionine ("M"), or leucine ("L"); X5 is a D- or L- isomer of the amino acid serine ("S") or asparagine ("N"); or a combination thereof. The composition of claim 1, further characterized in that the nucleic acid expression construct encodes a polypeptide of a sequence comprising SeqlD # 1, SeqlD # 2, SeqlD # 3, SeqlD # 4, or SeqlD # 5. 13. A composition comprising: (a) a nucleic acid expression construct; and (b) a poly-L-glutamate polypeptide associated therewith; characterized in that a mole ratio of the transfection facilitator loaded polypeptide with respect to the nucleic acid expression construct comprises from 1 mol to 5, 000 moles of the transfection facilitator loaded polypeptide per mole of nucleic acid expression construct. The composition of claim 13, further characterized in that the mole ratio of the transfection facilitator-loaded polypeptide to the nucleic acid expression construct is equal to 1,200 moles or less of the transfection facilitator-loaded polypeptide per mole of protein construction. nucleic acid expression 15. The composition of claim 13, further characterized in that the mole ratio of the transfection facilitator loaded polypeptide to the nucleic acid expression construct is equal to 1 mole of the transfection facilitator loaded polypeptide per mole. of nucleic acid expression construction. The composition of claim 13, further characterized in that an average molecular length of the nucleic acid expression vector is from about 2,000 to about 5,000 base pairs nucleotides. The composition of claim 13, further characterized in that an average molecular weight of the transfection facilitator loaded polypeptide is from about 400 to about 30,000 Da. 18. The composition of claim 13, further characterized in that an average molecular length of the nucleic acid expression vector is approximately 5,000 nucleotide base pairs, and an average molecular weight of the transfection facilitating transcription polypeptide is about 10,900 Da. The composition of claim 13, further characterized in that the nucleic acid expression construct comprises SeqlD # 11, SeqlD # 12, SeqlD # 13, SeqlD # 14, SeqlD # 17, SeqlD # 18, SeqlD # 19, SeqlD # 20 or SeqlD # 21. The composition of claim 13, further characterized in that the nucleic acid expression construct comprises a gene encoding a growth hormone releasing hormone ("GHRH") or its biological functional equivalent. The composition of claim 20, further characterized in that the encoded GHRH is a biologically active polypeptide, and the functional biological encoded equivalent of GHRH is a polypeptide that has been designed to contain a distinct amino acid sequence while simultaneously having similar or enhanced biological activity when compared to the GHRH polypeptide. 22. The composition of claim 20, further characterized in that the encoded GHRH or its functional biological equivalent is of the formula (SEQID # 6): -X-1-X2-DAIFTNSYRKVL-X3-QLSARKLLQDI-X4-X5-RQQGERNQEQGA-OH where the formula has the following characteristics: Xi is a D- or L- isomer of the amino acid tyrosine ("Y") or histidine ("H"); X2 is a D- or L- isomer of the amino acid alanine ("A"), valine ("V") or isoleucine ("I"); X3 is a D- or L- isomer of the amino acid alanine ("A") or glycine ("G"); X4 is a D- or L- isomer of the amino acid methionine ("M"), or leucine ("L"); X5 is a D- or L- isomer of the amino acid serine ("S") or. asparagine ("N"); or a combination thereof. The composition of claim 13, further characterized in that the nucleic acid expression construct encodes a polypeptide of a sequence comprising SeqlD # 1, SeqlD # 2, SeqlD # 3, SeqlD # 4, or SeqlD # 5. 24. A composition comprising: (a) a nucleic acid expression construct that encodes a growth hormone releasing hormone ("GHRH") or its functional biological equivalent; and (b) a poly-L-g lutamate polypeptide associated therewith; characterized in that a molar ratio of the transfection facilitating transcription polypeptide with respect to the nucleic acid expression construct comprises from 1 mole to 5,000 moles of the transfection facilitator loaded polypeptide per mole of nucleic acid expression construct. The composition of claim 24, further characterized in that the mole ratio of the transfected transfection facilitator polypeptide to the nucleic acid expression construct is equal to 1,200 mole or less of the transfection facilitator loaded polypeptide per mole of the nucleic acid expression. 26. The composition of claim 24, further characterized in that the mole ratio of the transfected transcription facilitating polypeptide to the nucleic acid expression construct is equal to 1 mole of the transfection facilitator loaded polypeptide per mole of expression construct. nucleic acid. The composition of claim 24, further characterized in that an average molecular length of the nucleic acid expression vector is from about 2,000 to about 5,000 base pairs nucleotides. The composition of claim 24, further characterized in that an average molecular weight of the transfection facilitating transcription polypeptide is from about 400 to about 30,000 Da. The composition of claim 24, further characterized in that an average molecular length of the nucleic acid expression vector is approximately 5,000 nucleotide base pairs, and an average molecular weight of the transfection facilitator loaded polypeptide is about 10.900 Da The composition of claim 24, further characterized in that the nucleic acid expression construct comprises SeqlD # 11, SeqlD # 12, SeqlD # 13, SeqlD # 14, SeqlD # 17, SeqlD # 18, SeqlD # 19, SeqlD # 20 or SeqlD # 21. The composition of claim 24, further characterized in that the encoded GHRH is a biologically active polypeptide, and the functional biological equivalent of encoded GHRH is a polypeptide that has been designed to contain a distinct amino acid sequence while simultaneously having similar or enhanced biological activity when compared to the GHRH polypeptide. 32. The composition of claim 24, further characterized in that the encoded GHRH or its functional biological equivalent is of the formula (SEQID # 6): -X- -X2-DAI FT SYRKVL-X3-QLSARKLLQDI-X4-X5-RQQGER QEQGA -OH where the formula has the following characteristics: Xt is a D- or L- isomer of the amino acid tyrosine ("Y") or histidine ("H"); X2 is a D- or L- isomer of the amino acid alanine ("A"), valine ("V") or isoleucine ("I"); X3 is a D- or L- isomer of the amino acid alanine ("A") or glycine ("G"); X4 is a D- or L- isomer of the amino acid methionine ("M"), or leucine ("L"); X5 is a D- or L- isomer of the amino acid serine ("S") or asparagine ("N"); or a combination thereof. 33. The composition of claim 24, further characterized in that the nucleic acid expression construct encodes a polypeptide of a sequence comprising SeqlD # 1, SeqlD # 2, SeqlD # 3, SeqlD # 4, or SeqlD # 5. 34. A composition comprising: (a) a nucleic acid expression construct that encodes a growth hormone releasing hormone ("GHRH") or its functional biological equivalent; and (b) a transfected transfection facilitator polypeptide associated therewith; characterized in that a molar ratio of the transfection facilitating transcription polypeptide with respect to the nucleic acid expression construct comprises from 1 mole to 5,000 moles of the transfection facilitator loaded polypeptide per mole of nucleic acid expression construct. 35. The composition of claim 34, further characterized in that the mole ratio of the transfection facilitating transcription polypeptide to the nucleic acid expression construct is equal to 1200 mole or less of the transfection facilitator loaded polypeptide per mole of the nucleic acid expression. 36. The composition of claim 34, further characterized in that the mole ratio of the transfected transcription facilitator polypeptide to the nucleic acid expression construct is equal to 1 mole of the transfection facilitator loaded polypeptide per mole of expression construct. nucleic acid. 37. The composition of claim 34, further characterized in that an average molecular length of the nucleic acid expression vector is from about 2,000 to about 5,000 base pairs nucleotides. 38. The composition of claim 34, further characterized in that an average molecular weight of the transfection facilitating transcription polypeptide is from about 400 to about 30,000 Da. 39. The composition of claim 34, further characterized in that an average molecular length of the nucleic acid expression vector is approximately 5,000 nucleotide base pairs, and an average molecular weight of the transfection facilitator loaded polypeptide is about 10.900 Da. 40. The composition of claim 34, further characterized in that the transfected transfection facilitator polypeptide comprises poly-L-glutamate. 41. The composition of claim 34, further characterized in that the nucleic acid expression construct comprises SeqlD # 11, SeqlD # 12, SeqlD # 13, SeqlD # 14, SeqlD # 17, SeqlD # 18, SeqlD # 19, SeqlD # 20 or SeqlD # 21. 42. The composition of claim 34, further characterized in that the encoded GHRH is a biologically active polypeptide, and the functional biological equivalent of encoded GHRH is a polypeptide that has been designed to contain a distinct amino acid sequence while simultaneously having similar or enhanced biological activity. when compared to the GHRH polypeptide. 43. The composition of claim 34, further characterized in that the encoded GHRH or its functional biological equivalent is of the formula (SEQID # 6): -X-1-X2-DAI FTNSYRKVL-X3-QLSARKLLQ D I-X4-X5- RQQGE RNQEQG A-OH wherein the formula has the following characteristics: X is a D- or L- isomer of the amino acid tyrosine ("Y") or histidine ("H"); X2 is a D- or L- isomer of the amino acid alanine ("A"), valine ("V") or isoleucine ("I"); X3 is a D- or L- isomer of the amino acid alanine ("A") or glycine ("G"); X4 is a D- or L- isomer of the amino acid methionine ("M"), or leucine ("L"); X5 is a D- or L- isomer of the amino acid serine ("S") or asparagine ("N"); or a combination thereof. 44. The composition of claim 34, further characterized in that the nucleic acid expression construct encodes a polypeptide of a sequence comprising SeqlD # 1, SeqlD # 2, SeqlD # 3, SeqlD # 4, or SeqlD # 5. 45. A method for introducing a nucleic acid expression construct into a cell of a selected tissue in a recipient, comprising: (a) placing a plurality of electrodes in the selected tissue, wherein the plurality of electrodes is disposed in a spaced relationship. (b) introducing the nucleic acid expression construct with a transfected transfection facilitator polypeptide associated therewith; and (c) applying an electric pulse of constant current to the plurality of electrodes; characterized in that a mole ratio of the transfection facilitating transcription polypeptide to the nucleic acid expression construct comprises from 1 mole to 5,000 moles of the transfection facilitator loaded polypeptide per mole of nucleic acid expression construct. 46. The composition of claim 45, further characterized in that the nucleic acid expression construct comprises SeqlD # 11, SeqlD # 12, SeqlD # 13, SeqlD # 14, SeqlD # 17, SeqlD # 18, SeqlD # 19, SeqlD # 20 or SeqlD # 21. 47. The method of claim 45, further characterized in that the cell of the selected tissue comprises a somatic cell, a stem cell, or a germ cell. 48. The method of claim 45, further characterized in that the tissue selected in the recipient comprises muscle. 49. The method of claim 45, further characterized in that the transfected transfection facilitator polypeptide comprises poly-L-glutamate. 50. The method of claim 45, further characterized in that the mole ratio of the transfection facilitator-loaded polypeptide to the nucleic acid expression construct is equal to 1,200 moles or less of the transfection facilitator-loaded polypeptide per mole of protein construction. nucleic acid expression. 51. The method of claim 45, further characterized in that the mole ratio of the transfection facilitator loaded polypeptide to the nucleic acid expression construct is equal to 1 mole of the transfection facilitator loaded polypeptide per mole of expression construct. nucleic acid. 52. The method of claim 45, further characterized in that the plurality of electrodes is constructed of a material that will make galvanic contact with the tissues. 53. The method of claim 45, further characterized in that the nucleic acid expression construct comprises a gene encoding a growth hormone releasing hormone ("GHRH") or its functional biological equivalent. 54. The method of claim 53, further characterized in that the encoded GHRH or its functional biological equivalent is expressed in a tissue-specific cell of the subject. 55. The method of claim 53, further characterized in that the encoded GHRH is a biologically active polypeptide, and the functional biological equivalent of encoded GHRH is a polypeptide that has been designed to contain a distinct amino acid sequence while simultaneously having similar or enhanced biological activity. when compared to the GHRH polypeptide. 56. The method of claim 53, further characterized in that the encoded GHRH or its functional biological equivalent is of the formula (SEQID # 6): -X- -X2-DAIFTNSYRKVL-X3-QLSARKLLQDI-X4-X5-RQQGERNQEQGA-OH in where the formula has the following characteristics: Xi is a D- or L- isomer of the amino acid tyrosine ("Y") or histidine ("H"); X2 is a D- or L- isomer of the amino acid alanine ("A"), valine ("V") or isoleucine ("I"); X3 is a D- or L- isomer of the amino acid alanine ("A") or glycine ("G"); X4 is a D- or L- isomer of the amino acid methionine ("M"), or leu ciña ("L"); X5 is a D- or L- isomer of the amino acid serine ("S") or asparagine ("N"); or a combination thereof. 57. The composition of claim 45, further characterized in that the nucleic acid expression construct encodes a polypeptide of a sequence comprising SeqlD # 1, SeqlD # 2, SeqlD # 3, SeqlD # 4, or SeqlD # 5. 58. A method for introducing a nucleic acid expression construct into a muscle cell in a body, comprising: (a) placing a plurality of electrodes on the selected tissue, wherein the plurality of electrodes is disposed in a spaced relationship. (b) introducing the nucleic acid expression construct with a transfection associated facilitator polypeptide associated therewith; wherein the transfection facilitator loaded polypeptide comprises a poly-L-glutamate polypeptide; (c) applying an electrical pulse to the plurality of electrodes; characterized in that the nucleic acid expression construct encodes a growth hormone releasing hormone ("GHRH") or its functional biological equivalent; and a mole ratio of the transfection facilitator loaded polypeptide to the nucleic acid expression construct comprises from 1 mole to 5,000 moles of the transfection facilitator loaded polypeptide per mole of nucleic acid expression construct. 59. The method of claim 58, further characterized in that an average molecular length of the nucleic acid expression vector is from about 2,000 to about 5,000 base pairs nucleotides. 60. The method of claim 58, further characterized in that an average molecular weight of the transfection facilitator loaded polypeptide is from about 400 to about 30,000 Da. 61. The method of claim 58, further characterized in that an average molecular length of the nucleic acid expression vector is about 5,000 nucleotide base pairs, and an average molecular weight of the transfection facilitator loaded polypeptide is about 10.900 Da. 62. The method of claim 58, further characterized in that the nucleic acid expression construct comprises SeqlD # 11, SeqlD # 12, SeqlD # 13, SeqlD # 14, SeqlD # 17, SeqlD # 18, SeqlD # 19, SeqlD # 20 or SeqlD # 21. 63. The method of claim 58, further characterized in that the mole ratio of the transfection facilitator-loaded polypeptide to the nucleic acid expression construct is equal to 1,200 moles or less of the transfection facilitator-loaded polypeptide per mole of protein construction. nucleic acid expression. 64. The method of claim 58, further characterized in that the mole ratio of the transfection facilitator-loaded polypeptide to the nucleic acid expression construct is equal to 1 mole of the transfection facilitator-loaded polypeptide per mole of expression construct. nucleic acid. 65. The method of claim 58, further characterized in that the plurality of electrodes is constructed of a material that will make galvanic contact with the tissues. 66. The method of claim 58, further characterized in that the introduction of the nucleic acid expression construct into the muscle cell of the recipient initiates the expression of a coded GHRH or its functional biological equivalent. 67. The method of claim 58, further characterized in that the encoded GHRH or its functional biological equivalent is expressed in a tissue-specific cell of the subject. 68. The method of claim 58, further characterized in that the encoded GHRH is a biologically active polypeptide, and the functional biological equivalent of encoded GHRH is a polypeptide that has been designed to contain a distinct amino acid sequence while simultaneously having similar or enhanced biological activity. when compared to the GHRH polypeptide. 69. The method of claim 58, further characterized in that the encoded GHRH or its functional biological equivalent is of the formula (SEQID # 6): -X-1-X2-DAIFT SYRKVL-X3-QLSARKLLQDI-X4-X5-RQQGERNQEQGA- OH wherein the formula has the following characteristics: Xi is a D- or L- isomer of the amino acid tyrosine ("Y") or histidine ("H"); X2 is a D- or L- isomer of the amino acid alanine ("A"), valine ("V") or isoleucine ("I"); X3 is a D- or L- isomer of the amino acid alanine ("A") or glycine ("G"); X4 is a D- or L- isomer of the amino acid methionine ("M"), or leucine ("L"); X5 is a D- or L- isomer of the amino acid serine ("S") or asparagine ("N"); or a combination thereof. 70. The method of claim 58, further characterized in that the nucleic acid expression construct encodes a polypeptide of a sequence comprising SeqlD # 1, SeqlD # 2, SeqlD # 3, SeqlD # 4, or SeqlD # 5. 71. A method for increasing the stability of a nucleic acid expression construct, comprising: mixing the nucleic acid expression construct with a transfection facilitator polypeptide to obtain a stabilized nucleic acid expression construct; characterized in that (a) the in vitro degradation of the nucleic acid expression construct is slower compared to that of the nucleic acid expression construct not associated with a transfection facilitator polypeptide; and (b) a molar ratio of the transfection facilitating transcription polypeptide with respect to the nucleic acid expression construct comprises from 1 mole to 5,000 moles of the transfection facilitator loaded polypeptide per mole of nucleic acid expression construct. 72. The method of claim 71, further characterized in that the transfection facilitator loaded polypeptide comprises a poly-L-glutamate polypeptide. 73. The method of claim 71, further characterized in that an average molecular length of the nucleic acid expression vector is from about 2,000 to about 5,000 base pairs nucleotides. 74. The method of claim 71, further characterized in that an average molecular weight of the transfection facilitator loaded polypeptide is from about 400 to about 30,000 Da. 75. The method of claim 71, further characterized in that an average molecular length of the nucleic acid expression vector is about 5,000 nucleotide base pairs, and an average molecular weight of the transfection facilitator loaded polypeptide is about 10.900 Da. 76. The method of claim 58, further characterized in that the nucleic acid expression construct comprises SeqlD # 11, SeqlD # 12, SeqlD # 13, SeqlD # 14, SeqlD # 17, SeqlD # 18, SeqlD # 19, SeqlD # 20 or SeqlD # 21. 77. The method of claim 71, further characterized in that the mole ratio of the transfection facilitator-loaded polypeptide to the nucleic acid expression construct is equal to 1,200 moles or less of the transfection facilitator-loaded polypeptide per mole of protein construction. nucleic acid expression. 78. The method of claim 71, further characterized in that a molar ratio of the transfected transfection facilitator polypeptide to the nucleic acid expression construct is equal to 1 mole of the transfection facilitator loaded polypeptide per mole of nucleic acid expression construct. 79. The method of claim 71 encodes a growth hormone releasing hormone ("GHRH") or its functional biological equivalent. 80. The method of claim 79, further characterized in that the encoded GHRH or its functional biological equivalent is of the formula (SEQID # 6): -X.1 -X2-D To the FT SYRKVL-X3-QLSARKLLQDI-X4-X5- RQQGERNQEQG A-OH where the formula has the following characteristics: Xi is a D- or L- isomer of the amino acid tyrosine ("Y") or histidine ("H"); X2 is a D- or L- isomer of the amino acid alanine ("A"), valine ("V") or isoleucine ("I"); X3 is a D- or L- isomer of the amino acid alanine ("A") or glycine ("G"); X4 is a D- or L- isomer of the amino acid methionine ("M"), or leucine ("L"); X5 is a D- or l_- isomer of the amino acid serine ("S") or asparagine ("N"); or a combination thereof. 81. The method of claim 71, further characterized in that the nucleic acid expression construct encodes a polypeptide of a sequence comprising SeqlD # 1, SeqlD # 2, SeqlD # 3, SeqlD # 4, or SeqlD # 5. 82. A method for increasing the stability of a nucleic acid expression construct, comprising: mixing the nucleic acid expression construct with a transfection facilitator polypeptide to obtain a stabilized nucleic acid expression construct; characterized in that the in vitro degradation of the nucleic acid expression construct is slower compared to that of the nucleic acid expression construct not associated with a transfection facilitator polypeptide; the transfection facilitator loaded polypeptide comprises a poly-L-glutamate polypeptide; the nucleic acid expression construct encodes a growth hormone releasing hormone ("GHRH") or its functional biological equivalent; and a molar ratio of the transfection facilitator loaded polypeptide to the nucleic acid expression construct comprises from 1 mole to 5,000 moles of the transfection facilitator loaded polypeptide per mole of nucleic acid expression construct. 83. The method of claim 8, further characterized in that an average molecular length of the nucleic acid expression vector is from about 2,000 to about 5,000 base pairs nucleotides. 84. The method of claim 82, further characterized in that an average molecular weight of the transfection facilitating transcription polypeptide is from about 400 to about 30,000 Da. 85. The method of claim 82, further characterized in that an average molecular length of the nucleic acid expression vector is about 5,000 base pairs nucleotides, and an average molecular weight of the transfection facilitator loaded polypeptide is about 10,900 Da. 86. The method of claim 82, further characterized in that the nucleic acid expression construct comprises SeqlD # 11, SeqlD # 12, SeqlD # 13, SeqlD # 14, SeqlD # 17, SeqlD # 18, SeqlD # 19, SeqlD # 20 or SeqlD # 21. 87. The method of claim 82, further characterized in that the mole ratio of the transfected transfection facilitator polypeptide to the nucleic acid expression construct is equal to 1200 mole or less of the transfection facilitator loaded polypeptide per mole of nucleic acid expression. 88. The method of claim 82, further characterized in that a mole ratio of the transfected transfection facilitator polypeptide to the nucleic acid expression construct is equal to 1 mole of the transfection facilitator loaded polypeptide per mole of expression construct. nucleic acid. 89. The method of claim 82, further characterized in that the encoded GHRH or its functional biological equivalent is of the formula (SEQID # 6): -Xi-Xa-DAIFTNSYRKVL-Xa-QLSARKLLQDI-Xi-Xs-RQQGERNQEQGA-OH where the formula has the following characteristics: Xi is a D- or L- isomer of the amino acid tyrosine ("Y") or histidine ("H"); X2 is a D- or L- isomer of the amino acid alanine ("A"), valine ("V") or isoleucine ("I"); X3 is a D- or L- isomer of the amino acid alanine ("A") or glycine ("G"); X4 is a D- or L- isomer of the amino acid methionine ("M"), or leucine ("L"); X5 is a D- or L- isomer of the amino acid serine ("S") or asparagine ("N"); or a combination thereof. 90. The method of claim 82, further characterized in that the nucleic acid expression construct encodes a polypeptide of a sequence comprising SeqlD # 1, SeqlD # 2, SeqlD # 3, SeqlD # 4, or 3eqlD # 5.