CN1662261A - Increased delivery of a nucleic acid construct in vivo by the poly-L-glutamate ( - Google Patents

Abstract

Plasmids delivered to muscle tissue by injection/electroporation can be expressed and physiological levels of the transgene can enter circulation. However, stabilization of naked DNA may be desirable, and in some cases even necessary, such as prolonged storage at different temperatures before use, injection into large numbers of animals, etc. It is essential that the compound of interest is not toxic to the cell, such as a muscle cell, or causes damage to the plasmid. It is preferred that the coated DNA is taken up at a similar or higher level by the cells of interest. Low molecular weight poly-L-glutamic acid compounds have all the desired properties. The molar/molar ratio of DNA/PLG is known to be the optimal concentration for gene therapy in skeletal muscle, resulting in increased expression of the transgene without causing damage to the target tissue. Furthermore, no one in the literature has observed or described the stabilization of plasmid DNA by PLG.

Description

Increased in vivo delivery of nucleic acid constructs by the poly-L-glutamic acid ("PLG") system

related application

[0001] This application is Serial No. 10/156,670, entitled "Plasmid-Mediated Gene Complementation and In Vivo Expression of the Poly-L-Glutamic Acid ("PLG") System", by Draghia- A continuation-in-part of US Patent Application filed 5/25/2002 by Akli et al., the entirety of which is hereby expressly incorporated by reference.

background

[0002] The delivery of isolated or recombinant proteins has been used for many years to correct a range of inborn or acquired defects and imbalances in individuals (eg, insulin for diabetes). Recently, nucleic acid expression constructs (ie, plasmids) with specific coding genes were delivered to bodily tissues and shown to be useful in correcting genetic defects. While both methods of protein replenishment work well, the method of replenishing nucleic acid expression constructs has several advantages over administration of recombinant proteins, such as conservation of native protein structure; enhanced biological activity; avoidance of systemic toxicity; and avoidance of infectious and toxic impurities. Furthermore, plasmid-mediated gene complementation methods allow individuals to be exposed to a longer therapeutic range of therapeutic proteins, as exemplified by the persistent levels of therapeutic proteins found in the individual's circulatory system.

[0003] The first limitation of the use of recombinant proteins is their limited bioavailability after each administration. In contrast, the bioavailability of plasmid-mediated gene complementation is not an issue because a single injection of plasmids into individual muscle tissue allows physiological expression over a broad period of time, as disclosed in WO 99/05300 and WO 01/06988 . Plasmid DNA constructs are attractive candidates for direct supplementation into individual skeletal muscles for therapy because plasmid DNA is a well-defined entity that is biochemically stable and has been used successfully for many years. Relatively low expression levels obtained after a single injection of plasmid DNA are sometimes sufficient to demonstrate biological activity of secreted peptides (Tsurumi et al., 1996). While not wishing to be bound by theory, injection of a plasmid construct can promote enzyme and hormone production in an individual in a manner that more closely mimics nature. Furthermore, direct injection of plasmid DNA into muscle tissue is simple, inexpensive, and safe in a non-viral way of replenishing gene products in vivo.

[0004] In contrast to viral vectors, plasmid-based expression systems can include artificial gene delivery systems in addition to nucleic acids encoding therapeutically effective gene products. In this way many of the dangers associated with viral vectors can be avoided. Because of the use of "species-specific" elements for gene delivery, plasmid (ie, non-viral expression system) products are generally less toxic, which minimizes the risk of immunogenicity often associated with viral vectors. To date there have been no reported cases of integration of plasmid vectors into the host chromosome, which minimizes the risk of adverse effects such as activation of oncogenes or inactivation of tumor suppressor genes during therapy. As an episomal system localized extrachromosomally, plasmids have well-defined pharmacokinetic and elimination profiles, resulting in a limited period of gene expression in the tissue of interest (Houk et al., 2001; Mahato et al., 1997).

[0005] Unfortunately, with many applications of plasmid-mediated gene complementation, the level of transgene expression is low due to the inefficiency of uptake of plasmid DNA by the therapeutic tissue (Wells et al., 1997). As a result, the use of direct injection of plasmid DNA into individuals for therapy has been limited in the past. For example, following a single direct injection, inefficient uptake of DNA by muscle fibers results in relatively low expression levels in normal, non-regenerative (Vitadello et al., 1994) or atrophic muscle (Takeshita et al., 1996). In addition, the period of transgene expression is short (Hartikka et al., 1996) (Danko and Wolff, 1994). Until recently, the most effective preclinical applications were limited to vaccines (Davis et al., 1994; Davis et al., 1993).

[0006] Extensive efforts have thus been made over the past 20 years to increase the delivery of plasmid DNA to cells by chemical and physical means (Danko et al., 1994). For example, chemical approaches such as lipofection/liposome fusion, polylysine concentrates with and without adenovirus promoters have been used with limited success (Fisher and Wilson, 1994). The use of specific compositions consisting of polyacrylic acid has been disclosed in International Patent Publication WO 94/24983. Naked DNA has been used as disclosed in International Patent Publication WO/11092. Additionally, physical means of plasmid delivery include electroporation, increased cell membrane permeability, and pressure. Although, each of these methods has had limited success, of all the methods listed, electroporation is the most promising.

[0007] While not wishing to be bound by theory, delivery of plasmid DNA into cells by electroporation involves the application of pulsed voltage electric fields to create transient pores on the surface of the cell membrane, thereby allowing the influx of exogenous plasmid DNA molecules (Smith and Nordstrom, 2000). By modulating the electrical pulses generated by the electroporation system, the efficiency with which nucleic acid molecules pass through the channels or pores can be modulated. US Patent No. 5,704,908 describes an electroporation apparatus for delivering molecules to cells in said location in a patient's body cavity. These pulsed voltage injection devices are also described in US Patents 5,439,440 and 5,702,304 and PCT WO 96/12520, 96/12006, 95/19805 and 97/07826.

[0008] Electroporation has previously been used to transfect tumor cells following plasmid injection (Nishi et al., 1997; Rols et al., 1998), or to deliver the antineoplastic drug bleomycin to skin and subcutaneous tumors (Belehradek et al. et al., 1994; Heller et al., 1996). Electroporation has also been used in rodents and other small animals, eg (Muramatsu et al., 1998; Aihara and Miyazaki, 1998; Hasegawa et al., 1998; Rizzuto et al. 1999). Modified intramuscular injection of plasmid DNA techniques following electroporation of skeletal muscle has been shown to result in high levels of circulating growth hormone releasing hormone ("GHRH") (Draghia-Akli et al., 1999) (Draghia-Akli et al., 2002b). In vivo electroporation of skeletal muscle allows plasmid DNA to be efficiently taken up by normal fibers and thus expressed. Electroporation is the use of an electric field to induce transient permeability of biological membrane pores and allow macromolecules, ions and water to pass from one side of the membrane to the other. Accordingly, electroporation has been used to introduce drugs, DNA or other molecules into multicellular tissues. This technique was first used in vivo to transfect tumor cells following injection of plasmid DNA (Rols et al., 1998), or to deliver the antineoplastic drug bleomycin to skin and subcutaneous tumors (Allegretti and Panje, 1994; Heller et al. People, 1996). Recently, numerous studies (mainly in small animals) have shown that this technique drastically increases plasmid uptake by skeletal muscle cells and allows production of therapeutic levels of peptides (Yasui et al. 2001; Yin and Tang, 2001). Previously, we reported that injection of myogenic expression vectors in mice and pigs delivered human growth hormone releasing hormone ("GHRH") cDNA into skeletal muscle and stimulated growth hormone ("GH") for at least two months Secretion (Draghia-Akli et al., 1997; Draghia-Akli et al., 1999).

[0009] Despite recent improvements in plasmid DNA transfer techniques, additional improvements in electroporation techniques and plasmid DNA compositions are still needed. For example, the electroporation method can theoretically be accomplished in a manner that does not cause permanent damage to the cells. In fact, the electroporation technique puts lethal stress on many cells and leads to the degradation of plasmid DNA (Hartikka et al., 2001). Furthermore, plasmids have heretofore been stored at lower temperatures than before use due to reduced stability and degradation (Evans et al., 2000).

[0010] We have now optimized a set of constant current electroporation delivery techniques and plasmid DNA compositions that prevent transitional cell damage and degradation of plasmid DNA during electroporation of plasmid DNA into muscle cells. For example, during electroporation, transfection-promoting polypeptides such as poly-L-glutamic acid ("PLG") will increase the uptake process. While not wishing to be bound by theory, some mechanisms of increased absorption may be exploited. For example, a transfection-promoting polypeptide can bind to the surface of a protein and promote uptake by increasing bioavailability, inhibiting the usual degradation processes in interstitial fluid (ie, protecting DNA from nucleases present in interstitial fluid). In cells, transfection-promoting polypeptides can prevent the delivery of DNA into lysosomes (ie, organelles in cells that degrade foreign DNA and/or proteins) by disrupting the assembly of microtubules (Fujii et al., 1986). While not wishing to be bound by theory, transfection-promoting polypeptides, such as PLG sets, occur naturally as attachments to protein side chains. Therefore transfection-promoting polypeptides have been used to increase the stability of anticancer drugs (Li et al., 2000), and as "glue" to seal wounds or prevent tissue bleeding during injury and tissue repair (Otani et al., 1998; Otani et al. People, 1996). Some transfection-promoting peptides, such as PLG, do not increase the immune response or produce antibodies. It should be emphasized that there is some evidence that certain transfection-promoting peptides are only effective when combined with the electroporation method. Furthermore, PLG has been shown to reduce muscle damage associated with plasmid delivery (Draghia-Akli et al., 2002a).

[0011] An efficient strategy for increasing the electroporation delivery of plasmid DNA constructs using transfection-promoting polypeptides and electroporation is described here and illustrated in skeletal muscle from three different mammals. The stability of the plasmids at elevated temperatures is also illustrated.

overview

[0012] One aspect of the invention is a composition that facilitates the electroporation of a nucleic acid expression construct that expresses a gene encoding it in the recipient into cells of a recipient. Compositions of the invention contain a nucleic acid expression construct associated with a charged transfection-facilitating polypeptide. The molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct in the preparation of the composition comprises from 1 to 5000 moles of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. In a preferred embodiment, the molar ratio is equal to 1 mole of nucleic acid expression construct to 1200 moles or less charged transfection-promoting polypeptide, in another preferred embodiment, the molar ratio is equal to 1 mole of nucleic acid expression construct to 1 mole of charged transfection-facilitating polypeptide. In a preferred embodiment, the transfection-facilitating polypeptide comprises a charged polypeptide (eg, poly-L-glutamic acid). In addition, the nucleic acid expression construct comprises SeqED#11, SeqID#12, SeqID#13, SeqID#14, SeqID#17, SeqID#18, SeqID#19, SeqID#20 or SeqID#21. In addition, the nucleic acid expression construct encodes growth hormone releasing hormone ("GHRH") or its biologically functional equivalent, such as HV-GHRH (SEQID #1), TI-GHRH (SEQID #2), TV-GHRH (SEQID #3) , 15/27/28-GHRH (SEQID #4), wt-GHRH (SEQID #5).

[0013] Another aspect of the invention is a method of introducing a nucleic acid expression construct into cells of a selected tissue in a recipient. The method comprises impaling a plurality of electrodes into a selected tissue, wherein the plurality of electrodes are arranged in a spatial relationship; introducing a composition comprising a nucleic acid expression construct associated with a charged transfection-facilitating polypeptide; and applying electrical pulses to the plurality of electrodes . However, caliper electrodes can also be used instead of needle electrodes. The molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct in the preparation of the composition comprises from 1 to 5000 moles of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. In a preferred embodiment, the molar ratio is equal to 1 mole of nucleic acid expression construct to 1200 moles or less charged transfection-promoting polypeptide, in another preferred embodiment, the molar ratio is equal to 1 mole of nucleic acid expression construct to 1 mole of charged transfection-facilitating polypeptide. In a preferred embodiment, the transfection-facilitating polypeptide comprises a charged polypeptide (eg, poly-L-glutamic acid). In addition, nucleic acid expression constructs include SeqED#11, SeqID#12, SeqID#13, SeqID#14, SeqID#17, SeqID#18, SeqID#19, SeqID#20 or SeqID#21. In addition, nucleic acid expression constructs encoding growth hormone releasing hormone ("GHRH") or its biologically functional equivalents are shown in HV-GHRH (SEQID #1), TI-GHRH (SEQID #2), TV-GHRH (SEQID #3) , 15/27/28-GHRH (SEQ ID #4), wt-GHRH (SEQ ID #5).

A third aspect of the present invention is a method for increasing the stability of a nucleic acid expression construct, comprising: mixing a nucleic acid expression construct with a charged transfection-promoting polypeptide, wherein the charged transfection-promoting polypeptide contains poly-L - glutamic acid polypeptides, and nucleic acid expression constructs for plasmid-mediated gene complementation. The method involves compositions prepared in a molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct comprising from 1 to 5000 moles of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. In a preferred embodiment, the molar ratio is equal to 1 mole of nucleic acid expression construct to 1200 moles or less charged transfection-promoting polypeptide, in another preferred embodiment, the molar ratio is equal to 1 mole of nucleic acid expression construct to 1 mole of charged transfection-facilitating polypeptide. In a preferred embodiment, the transfection-facilitating polypeptide comprises a charged polypeptide (eg, poly-L-glutamic acid). In addition, nucleic acid expression constructs include SeqED#11, SeqID#12, SeqID#13, SeqID#14, SeqID#17, SeqID#18, SeqID#19, SeqID#20 or SeqID#21. In addition, nucleic acid expression constructs encoding growth hormone releasing hormone ("GHRH") or its biologically functional equivalents are shown in HV-GHRH (SEQID #1), TI-GHRH (SEQID #2), TV-GHRH (SEQID #3) , 15/27/28-GHRH (SEQ ID #4), wt-GHRH (SEQ ID #5).

Brief description of the drawings

[0015] FIG. 1 shows an electrode arrangement of 6 electrodes in the form of 3 pairs of opposite electrodes used in the prior art. It further depicts the point of overlap of electroporation in a single set, which is the central point depicted by the asterisk graph;

[0016] Fig. 2 shows an electrode arrangement of 5 electrodes used in the present invention. It further describes how a needle-type symmetrical arrangement of electrodes without opposing electrode pairs produces a diverging pattern of electroporation events in regions where no congruent electroporation overlaps have developed, and the region of the diverging pattern resembles a pentagonal shape;

Fig. 3 has described the serum level of SEAP in the mouse of the expression plasmid pSP SEAP that is coated with the poly-L-glutamic acid of injecting various concentrations;

[0018] FIG. 4 depicts SEAP serum levels in pigs injected with expression plasmid pSP SEAP with or without poly-L-glutamic acid coating.

[0019] FIG. 5 depicts SEAP serum levels in dogs injected with expression plasmid pSP SEAP with or without poly-L-glutamate coating.

[0020] Figure 6 shows the increase in plasmid DNA stability in vitro when poly-L-glutamic acid was added to the solution. All samples were incubated at 37°C for 6 months.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

the term

[0021] The term "nucleic acid expression construct" as used herein refers to any type of genetic construct comprising an encoding RNA capable of being transcribed. The term "expression vector" can also be used alternatively.

[0022] The term "biologically functional equivalent" of GHRH, as used herein, is a polypeptide that has been engineered to have a different amino acid sequence when compared to a GHPH polypeptide, but at the same time have similar or improved biological activity.

[0023] The term "encoded GHRH" as used herein is a biologically active polypeptide.

[0024] The term "delivery" as used herein is defined as the introduction of a substance, with or without pressure, into an individual, cell or any recipient means.

[0025] The term "individual" as used herein refers to any species in the kingdom Animalia. In preferred embodiments it refers more specifically to humans and animals used as pets (such as cats, dogs, etc.), labor (such as horses, cows, etc.), food (such as chickens, fish, lambs, etc.), and animals that have been used in the art all other animals known.

[0026] The term "recipient" as used herein refers to any species in the kingdom Animalia. In preferred embodiments it refers more specifically to humans and animals used as pets (such as cats, dogs, etc.), labor (such as horses, cows, etc.), food (such as chickens, fish, lambs, etc.), and animals that have been used in the art all other animals known.

[0027] 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 conversely, a promoter can be constitutive so that an inducing agent cannot regulate the rate of transcription. Promoters can be regulated in a tissue-specific or tissue-preferred manner so that operably linked coding regions are transcribed only in particular tissue types.

[0028] The term "coding region" as used herein refers to any portion of a DNA sequence that is transcribed into messenger RNA ("mRNA") and subsequently translated into an amino acid sequence that characterizes a particular polypeptide.

The term "analogue" used herein includes any mutant of GHRH, or artificially synthesized or naturally occurring GHRH polypeptide fragments, such as HV-GHRH (SEQID #1), TI-GHRH (SEQID #2), TV- GHRH (SEQID#3), 15/27/28-GHRH (SEQID#4), (1-44) _NH2 or (1-40)OH (SEQID#6) forms or as short as (1-29)NH2 shorter form.

[0030] The term "somatotropin" ("GH") as used herein is defined as a growth-related hormone that acts as a chemical signal to target cells.

[0031] The term "growth hormone releasing hormone" ("GHRH") as used herein is defined as a hormone that promotes or stimulates the release of growth hormone, and to a lesser extent than other pituitary hormones such as lactoestrogens.

[0032] The term "molecular switch" as used herein refers to a molecule capable of regulating gene transcription upon entry into a subject.

[0033] The term "cassette" as used herein is defined as one or more transgene expression vectors.

The term "after 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 its biological equivalent to allow the expression of the encoded gene, while the modified cell is located in a living organism. in vivo.

[0035] The term "placement" as used herein refers to placement of multiple electrodes (which may be plate or needle electrodes) in selected tissue.

[0036] The term "heterologous nucleic acid sequence" as used herein is defined as a DNA sequence composed of distinct regulatory and expression elements.

[0037] The term "vector" as used herein refers to any means for delivering nucleic acid to a cell or organism. These include eg plasmids, viral vectors, liposomes or cationic lipids.

[0038] The term "electroporation" as used herein refers to a method of delivering nucleic acids to cells using electrical pulses.

[0039] The term "electrical pulse" as used herein refers to both a constant current pulse and a constant voltage pulse.

[0040] The term "poly-L-glutamic acid" as used herein refers to a biodegradable polymer of L-glutamic acid, the sodium salt of which, in some aspects of the invention, is suitable for use in Or a carrier or adjuvant that transfers DNA to cells without electroporation.

[0041] The term "spatial relationship" as used herein refers to the symmetrical or asymmetrical relationship of electrodes placed in the tissue of a subject to other electrodes.

[0042] The term "weight ratio" as used herein refers to the ratio of the amount (in milligrams) of the nucleic acid expression construct to the amount (in milligrams) of the charged transfection-facilitating polypeptide in the composition, regardless of the total volume delivered.

[0043] The term "molar ratio" as used herein refers to the ratio of the amount (in moles) of the nucleic acid expression construct to the amount (in moles) of the charged transfection-facilitating polypeptide in the composition.

[0044] The standard 1 and 3 letter shorthand for amino acids used here is as follows: alanine, A, ala; arginine, R, arg; asparagine, N, asn; aspartic acid, N, asp; Cystine, C, cys; Glutamine, Q, gln; Glutamic acid, E, glu; Glycine, G, gly; Histidine, H, his; Isoleucine, I, ile; Leucine , L, leu; Lysine, K, lys; Methionine, M, met; Phenylalanine, F, phe; Proline, P, pro; Serine, S, ser; Threonine, T , thr; tryptophan, W, trp; tyrosine, Y, tyr; valine, V, val.

[0045] The ability of electroporation to promote plasmid uptake by skeletal muscle has been well documented. However, the effective composition of nucleic acid expression vectors and transfection-promoting reagents for electroporation protocols is not described in the literature. The present invention describes compositions and methods for increasing the delivery of nucleic acid expression constructs to a recipient.

[0046] Composition Formulation: As described above, the ability of electroporation to promote plasmid uptake by skeletal muscle has been well documented. Other methods that do not involve electroporation have also been shown to increase plasmid uptake, for example, in the form of transfection-promoting particles—poly-L-glutamic acid ("PLG") or polyvinylpyrrolidone ("PVP") preparations have been tested. Increased gene transfection and consequently up to 10-fold increase in gene expression in muscle of mice, rats and dogs was observed. One aspect of the invention is the combination of electroporation and transfection-promoting particles associated with nucleic acid expression vectors. While not wanting to be bound by theory, PLG will increase plasmid transfection during electroporation, not only by physically stabilizing plasmid DNA and facilitating intracellular transport through membrane pores, but also by activating the transport machinery. For example, positively charged cell surface proteins attract and form complexes with negatively charged PLG associated with plasmid DNA through protein-protein interactions. When an electric field is applied, the surface proteins reverse direction and actively drive the DNA molecules inside. Furthermore, PLG/DNA molecules in contact with the cell membrane surface only need to move across the plasma membrane, whereas DNA molecules in the interstitium far from the cell surface are disadvantaged. Thus, protein-protein interactions, and proximity to transfection particles can substantially increase transfection efficiency.

[0047] Poly-L-glutamic acid ("PLG") is a stable compound that is resistant to high temperature denaturing conditions. PLG has previously been used to increase the stability of vaccine formulations as it does not increase the immunogenicity of the vaccine. In addition, PLG has been used as an antiviral agent after antigen inhalation or exposure to extreme ozone. Plasmid DNA delivered into skeletal muscle by injection, electroporation, or both is readily expressed and can be measured by the circulating physiological levels of the transgene product. However, stabilization of naked DNA is desirable, or even necessary, in some cases, such as prolonged storage prior to use, injection into large numbers of animals. Because plasmid DNA may be stored at different temperatures for different periods of time, a long-term stable plasmid solution is critical. It is important that the compound associated with the plasmid is not toxic to the cell (eg muscle cells) and does not cause damage to the plasmid DNA. It is preferred that the combination of plasmid DNA and associated transfection-facilitating particles is taken up similarly or increased by target cells. The present invention utilizes low or medium molecular weight poly-L-glutamic acid (such as 1-15kDa, average molecular weight is 10KDa or 15-50kDa, The average molecular weight is 35kDa) compounds exhibit all the properties required for effective nucleic acid expression vectors and transfection-promoting polypeptide compositions. Although PLG can be used at high concentrations in non-electroporation applications, we determined that a low molar ratio of nucleic acid expression vector to PLG is optimal for electroporation applications targeting skeletal muscle. An example of a useful molar ratio of nucleic acid expression vector to PLG is about 1:5000. Another example of a more useful nucleic acid expression vector to PLG molar ratio comprises about 1:2500. An example of a preferred molar ratio of nucleic acid expression vector to PLG is about 1:1200. An exemplary nucleic acid expression vector to PLG molar ratio comprises about 1:800. A representative nucleic acid expression vector to PLG molar ratio comprises about 1:500. In one example, the molar ratio of nucleic acid expression vector to PLG selected comprises about 1:200. In another example, the more preferred molar ratio of nucleic acid expression vector to PLG is about 1:100. An example of a preferred molar ratio of nucleic acid expression vector to PLG comprises about 1:50. Included in another more preferred molar ratio of nucleic acid expression vector to PLG is about 1:20. In an even more preferred example the molar ratio of nucleic acid expression vector to PLG comprises about 1:10. In a most preferred example, the molar ratio of nucleic acid expression vector to PLG is about 1:1.

According to the number of moles of the nucleic acid expression carrier (such as the scope of 2,000bp to 30,000bp) of appropriate average length to low or medium molecular weight poly-L-glutamic acid (such as 1-15kDa, average molecular weight is 10KDa or 15 -50kDa, the average molecular weight is 35kDa) of PLG moles, can calculate the appropriate molar ratio. Electroporation of the plasmid DNA composition in combination with PLG resulted in increased expression of the reporter transgene without damage to the target tissue.

[0049] Therefore, the pharmaceutical composition of the present invention can be delivered to various locations in the animal body via various routes to achieve special effects. Those skilled in the art recognize that although more than one route may be used to administer a drug, a particular route may provide a more immediate and effective response than others. While not wishing to be bound by theory, the use of a composition formulated for application or placement into a body cavity, inhalation or spray aerosol, or parenteral administration including intramuscular, intravenous, peritoneal, subcutaneous, intradermal, and topical Introduction can be accomplished for local or systemic delivery. Thus, different delivery methods can be used to deliver the plasmid/promoter composition into cells. Examples include: (1) methods utilizing physical means such as electroporation (electricity), gene guns (physical force) or the application of large volumes of fluid (pressure); and (2) the combination of the carrier with other entities (such as liposomes) or carrier molecules) to form complexes.

[0050] Constant Current Electroporation: The essential phenomenon of electroporation is believed to be the same in all cases, but the exact mechanism underlying the observed effects has not been clarified. While not wishing to be bound by theory, a well-publicized indication of the effect of electroporation is that the cell membrane becomes transiently permeable to macromolecules after the cells are exposed to an electrical pulse. Under normal circumstances, there are channels running through the cell wall, which maintain a static transmembrane voltage of ca.90mV by allowing bidirectional movement of ions.

[0051] While not wishing to be bound by theory, electroporation utilizes the same structures by forcing a high flow of ions through them, thereby opening or enlarging channels. In the prior art, metal electrodes are placed in contact with the tissue, and a predetermined voltage is applied to the electrodes in proportion to the distance between the electrodes. Protocols for electroporation are defined in terms of the resulting electric field strength, according to the general formula E=V/d, where ("E") is the electric field, ("V') is the applied voltage, and ("d") is the distance.

[0052] In the prior art, the electric field strength E is a very important value when formulating an electroporation protocol for delivering drugs or macromolecules into the cells of a subject. Therefore, any electric field strength for various scenarios can be calculated by applying preset voltage pulses proportional to the distance between the electrodes. However, there is a caveat that electric fields can also be generated in tissue using insulated electrodes (ie flow of ions is not necessary to create electric fields). While not wanting to be bound by theory, it is the electrical current rather than the electric field itself that is necessary for successful electroporation.

[0053] In the process of electroporation, the heat generated is the product of the resistance between electrodes, the square of the current and the duration of the pulse. During electroporation, heat is generated in the tissue, which can be thought of as the product of the current across the electrodes, the voltage, and the duration of the pulse. The currently described electroporation protocol, defined in terms of the resulting electric field strength E, relies on short voltage pulses of unknown current. Therefore, the resistance and heat generated by the tissue cannot be measured, which leads to differences in the success rate of different pulsed voltage electroporation protocols using predetermined voltages. The ability to limit cellular heat generation across the electrodes can increase the effectiveness of any given electroporation voltage pulse protocol.

[0054] Controlling the electrical current across the electrodes allows one to determine the relative heat production of the cells. Thus, it is the current rather than the voltage across the electrodes that determines the subsequent effect of any given pulse regimen. Predetermined voltages do not produce predetermined currents, and the prior art does not provide a means to accurately measure current dose, which limits the utility of this technology. Therefore, compared with pulses of predetermined voltages, controlling the current flow in tissue between two electrodes at a certain threshold will allow one to change the pulse conditions, reduce cell heating, reduce cell death, and more efficiently integrate macromolecules into cells. .

[0055] The constant current electroporation device was filed on March 7, 2002, with Westerstein et al. ("the Western '362 application") as the inventor, entitled "Electrodes Assembled and Use for Constant Current Electroporation" Invention of co-pending application S/N 60/362,362, drafted for incorporation by reference. One aspect of the Western '362 application overcomes this difficulty by precisely controlling the flow of ions that invade cell membrane channels, thus providing a means to effectively control the amount of electricity delivered to the cell within the range between the electrodes. Thus, the precise charge to the tissue can be calculated as the product of the delivered current level, pulse length and number of pulses. The constant current system includes an electrode arrangement connected to a specially designed circuit, this electrode is also utilized by the present invention.

[0056] One aspect of the present invention provides a means of delivering electroporation current along multiple channels to a volume of tissue without causing excessively concentrated cumulative current in any locality, thus avoiding cell death due to tissue overheating. The nucleic acid expression vector composition associated with the transfection-promoting polypeptide will further promote the success of the transfection protocol. For example, the maximum energy delivery for a particular pulse will occur along the line connecting the two electrodes. The prior art assumes that electrodes should be present in pairs, and that voltage pulses are delivered to pairs of electrodes of opposite polarity. Thus, the maximum energy delivery for a given pulse will occur along the line connecting the two electrodes. An example of an energy delivery path for prior art electrodes (using three pairs of radial electrodes and a central electrode) is described above and shown in FIG. 1 . The distribution of energy is staggered at the center point of the electrodes, which causes unnecessary heat generation and reduces cell viability. Thus, the nucleic acid/transfection-facilitating compositions of the present invention can also help stabilize cells in prior art electroporation protocols.

[0057] The electrodes of one embodiment of the present invention are arranged in a radial and symmetrical manner, but unlike the prior art, the electrodes are odd and not in opposing pairs (FIG. 2). Delivery of electrical pulses from an electrical pulse generator to any two electrodes produces a pattern that is best described as a polygon. The trajectory of this pattern is a pentagram with pentagonal electrical pulses around the center of the arrangement where the concentration of the transfected molecule is highest in the tissue. While not wanting to be bound by theory, it's not the odd number of electrodes per se that makes the difference, but rather the orientation of the circuit, using state of the art configurations, all pulses produce current through the center of the rig. Cumulative dose, and thermal effects, are thus concentrated centrally, while peripheral dose falls rapidly. Using the "pentagram arrangement", the dose is spread more evenly across a larger volume. For example, if the electrodes are arranged in a five-electrode formation, pulses may be applied to electrodes 1 and 3, then 3 and 5, then 5 and 2, then 2 and 4, then 4 and 1, in succession. However, because the tissue between the electrodes is a volume wire, a certain current intensity exists along the parallel lines, and the intensity decreases as the distance along the centerline increases. The cumulative effect of a series of pulses results in a more consistent spread of energy delivered to the tissue, increasing the likelihood that the cells being electroporated will actually survive the process.

[0058] It is known in the art that the nature of the voltage pulse to be generated depends on the nature of the tissue, the size of the selected tissue and the distance between the electrodes. What is required is that the electrical pulses be as uniform as possible and of the correct amplitude. Excessive electric field strengths can lead to cell lysis, while low electric field strengths can reduce the efficiency of electroporation. Prior art inventions use the distance between the electrodes to calculate the electric field strength and predetermine the voltage pulse for electroporation. This reliance on known inter-electrode distances is a limitation on electrode design. Since the programmable current pulse controller will determine the resistance of the two electrodes in a given volume of tissue, the distance between the electrodes is not a critical factor in determining the appropriate current pulse. Therefore, an alternative embodiment of a needle electrode arrangement would be an asymmetric arrangement of electrodes. Additionally, many suitable symmetric and asymmetric needle electrode arrangements can be envisioned by those skilled in the art without departing from the spirit and scope of the particular electrode design. The depth of each individual electrode in the array located in the tissue of interest can be varied based on comparable results. In addition, multipoint injection of macromolecules can be incorporated into needle-type electrode arrays.

[0059] By utilizing the constant current electroporation device described in the Western '362 application, a simple means of measuring the temperature rise of tissue exposed to the pulses can be obtained. For example, the measured interelectrode resistance times the current squared and the pulse time is a measure of the total energy delivered. When the volume of tissue surrounded by the electrodes and the specific heat of the tissue are known, the energy delivered can be converted to degrees Celsius. The rise in tissue temperature ("T", degrees Celsius) is the conversion factor between resistance ("R", ohms), current ("I", amperes), pulse length ("t", seconds) and joules to calories ("K"). T = RI ² tK.

[0060] During electroporation, the increase in current in prior art systems when a predetermined voltage is applied to the electrodes is due to the increase in cell permeability which reduces the resistance between the electrodes. This causes a transitional temperature rise, with cell death as a result. For example, using the usual values for conventional electroporation, assuming a volume surrounded by electrodes of 1 cubic centimeter, the tissue specific heat is close to uniform, with a temperature rise resulting from a 50 millisecond pulse of an average 5 amp current through a typically 25 ohm load resistance It is ca7.5°C. This points to the necessity of inserting a sufficient delay between successive pulses to allow sufficient heat removal by the subject's circulatory system so that the cumulative temperature rise does not result in destruction of the electroporated tissue.

[0061] The advantage of constant current is that it prevents the pulse from reaching a cell-damaging amplitude. In systems at predetermined voltages, the current may reach destructive levels without the operator being able to prevent it from happening. In a constant current system, the current is placed at a threshold level that does not result in cell death. The proper setting of the current depends on the electrode structure and must be determined experimentally. But once the appropriate levels are established, cell survival from case to case is guaranteed. Addition of a nucleic acid expression construct in conjunction with a transfection-enhancing polypeptide increases the chances of electroporated cells integrating the plasmid construct.

[0062] Nucleic Acid Constructs for Therapy: One aspect of the present invention relates to compositions and methods for the effective delivery of nucleic acid constructs to tissues as a means of treating various diseases in chronically ill subjects. Aspects of the invention relate to methods of delivering heterologous nucleic acid sequences encoding specific genes (such as growth hormone releasing hormone ("GHRH") into one or more cells of a subject (such as somatic, stem, or germ cells). ") or its biological equivalent), and when the modified cell is present in the individual, it allows the expression of the encoding gene (such as GHRH or its biological equivalent). A method of delivering the nucleic acid sequence encoding the gene is by electroporation. Subsequent expression of the encoded gene can be regulated by tissue-specific promoters (e.g., muscle), and/or regulatory proteins containing modified ligand-binding domains (e.g., molecular switches), which are only regulated by the correct modified ligand. It is activated when the body (such as mifepristone) is added to the individual from the outside. For example, extracranial expression and subsequent release of GHRH or its biological equivalents by modified cells can be used to treat anemia, wasting, immune dysfunction or other diseases, and prolong lifespan in chronically ill individuals.

[0063] Recombinant GH replacement therapy is widely used clinically with beneficial effects, but the usual dose is physiologically excessive. Such elevated recombinant GH doses are associated with deleterious side effects, eg, increased frequency of insulin resistance reported in up to 30% of recombinant GH treated patients, or accelerated epiphyseal growth and closure in pediatric patients. Additionally, the molecular heterogeneity of circulating GH may have important implications in growth and homeostasis, which may lead to a reduced potential of GH with a reduced ability to stimulate prolactin recipients. These adverse side effects are due to the use of recombinant exogenous GH protein to increase basal levels of GH and abolish the GH bursts. In contrast, no side effects have been reported for the treatment of recombinant GHRH. Normal levels of GHRH in the pituitary portal range from 150 to 800 pg/ml, while systemic circulating values for this hormone are 100-500 pg/ml. Some patients with acromegaly caused by extracranial tumors had nearly 100-fold higher levels (eg, 50 ng/ml of immunoreactive GHRH). Long-term studies (1-5 years) of recombinant GHRH therapy in children and elderly patients have shown no classic GH side effects, such as changes in fasting glucose concentrations or accelerated epiphyseal growth and closure of the femoral epiphysis in pediatric patients or Slip. Therefore, treatment with recombinant GHRH may be closer to physiological conditions than treatment with recombinant GH. Unfortunately, due to the short half-life of GHRH peptides in vivo, frequent intravenous or subcutaneous injections (ie, 1 to 3 times per day) are required if recombinant proteins are used. Gene transfer methods can overcome this limitation on the use of GHRH. In addition, a wide range of doses can exert therapeutic effects. The fact that the GHRH gene, cDNA, and native and some mutant molecules have been characterized in pigs and some other species, and that measurement of gene therapy efficacy is straightforward, support the selection of GHRH for gene therapy applications.

[0064] The invention may be better understood with reference to the following examples, which represent some embodiments of the invention and are not intended to limit the invention.

[0065] Example 1. A plasmid containing the muscle-specific artificial promoter SPc5-12 was previously described (Li et al., 1999). Wild-type and mutated porcine GHRH cDNAs were generated by GHRH cDNA site-directed mutagenesis (AlteredSites II in vitro Mutagenesis System, Promega, Madison, WI) and cloned into the BamH I/Hind III sites of pSPc5-12 to generate pSPc5-12, respectively - wt-GHRH or pSP-HV-GHRH. The 3' untranslated region (3'UTR) of growth hormone was cloned downstream of the GHRH cDNA. The resulting plasmid contains a mutated GHRH coding region, resulting in an amino acid sequence that does not naturally occur in mammalian cells. While not wishing to be bound by theory, treatment of anemia; increase of total red blood cell mass in an individual; reversal of wasting; reversal of abnormal weight loss; treatment of immune dysfunction; reversal of inhibition of lymphocyte proliferation; Effects are ultimately measured by circulating levels of GHRH hormone analogs. Some of the different plasmids encoding different mutated amino acid sequences of GHRH or its biologically functional equivalents are as follows:

Plasmid Encoded Amino Acid Sequence

wt-GHRH YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGERNQEQ GA-OH (SEQID#5)

HV-GHRH HVDAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQ GA-OH (SEQID#1)

TI-GHRH YIDAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQ GA-OH (SEQID#2)

TV-GHRH YVDAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQ GA-OH (SEQID#3)

15/27/28-GHRH YADAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQGHRH GA-OH (SEQID#4)

Typically, the encoded GHRH or its biologically functional equivalent is of the general formula (SeqID #6):

-A ₁ -A ₂ -DAIFTNSYRKVL-A ₃ -QLSARKLLQDI-A ₄ -A ₅ -RQQGERNQEQGA-OH, where standard 1-letter amino acid abbreviations are used; A1 is selected from tyrosine ("Y") or histidine ( "H") the amino acid D- or L-isomer; A2 is the amino acid D- selected from alanine ("A"), valine ("V") or isoleucine ("I") or L-isomer; A3 is an amino acid D- or L-isomer selected from alanine ("A") or glycine ("G"); A4 is selected from methionine ("M") or the amino acid D- or L-isomer of leucine ("L"); A5 is the amino acid D- or L-isomer selected from serine ("S") or asparagine ("N").

[0066] Other plasmids utilized include the pSP-SEAP construct containing the Sacl/HindIII SPc5-12 fragment, the SEAP gene, and the SV40 3'UTR from the pSEAP-2Basic vector (Clontech Laboratories, Inc., Palo Alto, CA).

The plasmid described above does not contain polylinker, IGF-1 gene, skeletal muscle α-actin promoter or skeletal muscle α-actin 3' UTR (untranslated region)/NCR (non-coding region) . In addition, these plasmids were introduced by intramuscular injection followed by in vivo electroporation as described below.

The term "biological function equivalent" is well known to those skilled in the art, and the inherent definition of "biological function equivalent" protein and/or polypeptide is that the molecule after the change will have an acceptable biological equivalent activity level, so in the molecular The number of changes that can occur in a given site is limited. Thus, biologically functional equivalents are defined herein as proteins (and polypeptides) in which selected amino acids (or codons) may be substituted. A peptide comprising a biologically functional equivalent of GHRH is a polypeptide engineered to have a different amino acid sequence compared to GHRH, while having similar or improved biological activity. For example, one biological activity of GHRH promotes the secretion of growth hormone ("GH") in an individual.

[0069] Plasmids associated with PLG in mice. To demonstrate that nucleic acid expression constructs associated with transfection-promoting polypeptides facilitate uptake by electroporated cells, a series of electroporation experiments were designed. Three separate sets of experiments were performed on mice. All mice were administered a total of 30 μg (micrograms) of pSP-SEAP (approximately 5000 base pairs ("bp")), +/- PLG (average molecular weight MW = 10900) in a total volume of 25 μl. One group of 10 mice received uncoated naked plasmids; subsequent groups received PLG-coated plasmids with sequentially decreasing PLG concentrations (see Table 1).

Group Total injection volume (μl) DNA (μg) PLG (μg/μl) Total PLG (μg) Approximate molar ratio of DNA:PLG 1 25 30 0.00 0.00 - 2 25 30 6.00 150 1:1200 3 25 30 1.00 25 1:200 4 25 30 0.10 2.50 1:20 5 25 30 0.01 0.25 1:2

Molar ratios are provided as examples. The molar ratios listed in Table 1 are based on a 5000bp nucleic acid expression vector and PLG with an average molecular weight of 10900. For example, group 2 in Table 1 was injected with a total of 30 μg of DNA vector combined with 150 μg of transfection-promoting polypeptide, wherein the molar ratio was 1:1200. Another example is that group 3 in Table 1 was injected with a total of 30 μg of DNA vector combined with 0.25 μg of transfection-promoting polypeptide, wherein the molar ratio was 1:2. Molar ratios of DNA vector to PLG with a 1:1 relationship (including lower restrictive formulations) still yielded higher transfection efficiencies than "naked" DNA vector alone. Ordinary skills in the art can formulate expression vectors of different lengths and calculate the molar ratio of PLG with various molecular weights. In addition, the length of known nucleic acid expression vector and the weighted average molecular weight of PLG can change based on the specific formulation strategy of specific vector length and those skilled in the art (such as functional nucleic acid expression vector is more or less than 5000 nucleosides, PLG has average molecular weight of less than about 1 to about 30 KDa). Therefore, even the smallest PLG polymers, such as PLG trimers with a molecular weight of about 400 Da, can be used in the present invention.

[0070] Electroporation was performed using a constant current electroporator, which is the subject of co-pending application Western '362. This device was used to deliver square wave pulses in all experiments. An amplitude condition of 1 mA, 5 pulses, 50 msec per pulse was used. Caliper electrodes are used to deliver electrical pulses in vivo. The caliper (plate) electrode consists of a 1.5 cm2 metal plate mounted on a scale so that the distance between the parallel plates can be easily assessed. Plasmid DNA or related DNA was injected through intact skin into the tibialis anterior muscle of mice. Each animal received one injection at one injection site. Although a constant current electroporation device was used in a specific example, it does not limit the general practice of the invention (ie, other electroporation devices may also provide satisfactory results). Furthermore, the order of electrode placement and subsequent plasmid injection is not sequence-restricted.

[0071] In order to determine the expression level of the SEAP gene product encoded by the DNA vector, the mice were bled 3 months after the injection of the plasmid and the serum was collected. SEAP usually disappears after birth and is immunogenic in adult animals. Mice blood was collected from the tail vein before plasmid administration and up to 3 months after injection. SEAP serum levels were determined using a chemiluminescent assay (Tropix, Bedford, MA) following the manufacturer's instructions. Figure 3 represents the serum SEAP levels of all five groups of mice described in Table 1. While naked plasmids (group 1, Figure 3) showed some expression, all groups using nucleic acid expression vectors in combination with PLG (groups 2-5, Figure 3) showed significantly higher serum SEAP levels. However, when the inflammatory markers (such as macrophages, B-cells, and counterstained with hematoxylin/eosin) of selected samples from each group were analyzed by immunohistochemistry, mice from group 5 (ie, treated with 0.01 μg/ μl PLG-coated nucleic acid expression construct) had the least inflammation associated with the delivery process 3 days after injection. Group 2 injected with plasmids combined with 6 μg/μl PLG had higher inflammation and some morphological changes despite higher expression in the early stage. This observation is consistent with data in the literature showing a short-term increase in expression following administration of PLG compounds, with expression disappearing approximately one month after injection (Fewell et al., 2001).

[0072] Tissue Analysis - Muscle and skin samples were fixed overnight, dehydrated in alcohol and embedded in paraffin. 5 μm sections were cut and stained with hematoxylin/eosin (Sigma Chemical, St. Louis, MO). Serial sections were stained with picric acid. Digital images of sections were captured using a CoolSnap digital color camera (Roper Scientific, Tucson, AZ) with MetaMorph software (Universal Imaging Corporation, Downington, PA) and a Zeiss Axioplan 2 microscope with a 40x objective (0.75 plan objective aperture).

[0073] Statistics Use STATISTICA analysis software package (StatSoft, Inc. Tulsa, OK) to carry out statistical analysis of data. The values represented in the graphs are mean ± s.e.m. Specific P values were obtained by comparison using ANOVA. P<0.05 was set as a significant difference level.

[0074] Example 2 - PLG coating experiment with pigs: In order to show similar results in larger mammals, an experiment similar to that of Example 1 above was carried out with pigs as the experimental subjects. Therefore, two groups of 3 pigs were injected with 500 μg (micrograms) of pSP-SEAP and electroporated. The plasmid expresses secreted embryonic alkaline phosphatase ("SEAP"). This molecule usually disappears after birth and is immunogenic in adult animals. One group received naked nucleic acid constructs and the other group received nucleic acid constructs in 0.01 μg/μl PLG. Pigs were weighed and bled every other day prior to injection and until 10 days post-injection. Pig serum was collected by jugular vein puncture before injection and at 2, 4, 6, 8 and 10 days after injection. SEAP serum levels were determined using a chemiluminescent assay (Tropix, Bedford, MA) following the manufacturer's instructions. SEPA analysis (FIG. 4) indicated that injection of PLG-coated plasmids in animals had increased expression relative to naked plasmids throughout the 12 days of the experiment (32.9 ± 19.3 ng/ml/kg of PLG/plasmid-pig vs. injection of naked 17.14 ± 12.44 ng/ml/kg for plasmid animals). While not wishing to be bound by theory, the increased expression may be due to increased plasmid stability, or facilitated transfection into muscle cells, or both.

[0075] Electroporation Apparatus A constant current electroporator machine (Advisys, Inc.) was used to deliver square wave pulses in all experiments. Electroporation parameters included an amplitude condition of 1 mA, 5 pulses, 50 msec per pulse. Needle electrodes are used to deliver electrical pulses inside the body. The 5-pin electrode assembly consisted of an equally spaced circular array of 21-gauge needles (1 cm diameter) mounted on insulating material. All needles are 2 cm long, and in all injections or electroporations, the needles are fully inserted into the muscle. Plasmid DNA was injected through intact skin using a 21 g needle into the porcine half-key muscle. Each animal received a single site injection and the injection site was tattooed for easy separation after the experiment.

[0076] Tissue Analysis - Muscle and skin samples were fixed overnight, dehydrated in alcohol and embedded in paraffin. 5 μm sections were cut and stained with hematoxylin/eosin (Sigma Chemical, St. Louis, MO). Serial sections were stained with picric acid. Digital images of sections were captured using a CoolSnap digital color camera (Roper Scientific, Tucson, AZ) with MetaMorph software (Universal Imaging Corporation, Downington, PA) and a Zeiss Axioplan 2 microscope with a 40x objective (0.75 plan objective aperture).

[0077] Statistics Use STATISTICA analysis software package (StatSoft, Inc. Tulsa, OK) to carry out statistical analysis of data. The values represented in the graphs are mean ± s.e.m. Specific P values were obtained by comparison using ANOVA. P<0.05 was set as a significant difference level.

[0078] Example 3 - PLG coating experiment with dogs: In order to show similar results in larger mammals of different species, an experiment similar to that of Example 2 above was carried out with dogs as subjects. Therefore, expression comparisons using 5-pin array electrode injection of coated or naked plasmids were performed in dogs. A total of 4 groups of 5 dogs were injected with pSP-SEAP plasmid expressing secreted embryonic alkaline phosphatase ("SEAP"). This molecule usually disappears after birth and is immunogenic in adult animals. There were no adverse reactions, or changes in biochemical, clinical or hormonal characteristics, associated with the development of an immune response against SEAP in the animals. Electroporation using standard conditions and 5-needle electrodes after injection was performed as described above. Plasmid DNA was either naked or coated with poly-L-glutamate mol/mol dilutions. The situation of each group is as follows:

Groups 1-5 needles (5N), 0.5mg, naked (NK)

Group 2-5 needles (5N), 0.1mg, naked (NK)

Group 3-5 needles (5N), 0.5 mg, coated (PLG)

Group 4-5 needles (5N), 0.1 mg, coated (PLG)

[0079] Dogs were weighed and bled every other day at baseline (pre-injection) and up to 10 days post-injection. Serum was used for SEPA analysis. Values are corrected by weight (blood volume). The differences in SEAP values between different injection groups were analyzed. The results of these experiments are shown in Figure 5. The results indicate that 5-pin electrodes can be used to efficiently mediate electroporation in dogs. In addition, PLG-coated DNA can increase plasmid stability and electroporation efficiency in dogs.

[0080] Example 4: PLG increases plasmid stability at high temperature in vitro: In order to evaluate the effect of PLG on plasmid stability, the following analysis was performed. The pSP-HV-GHRH plasmid encoding super-porcine growth hormone releasing hormone was diluted with distilled water to a final concentration of 2 mg/ml. A mol/mol ratio of PLG was added to the sample group but not to the control group. All samples were incubated at 37°C for 6 months. After six months, an aliquot of all samples was taken for agarose gel electrophoresis (Fig. 6). As seen in the gel images, all plasmids were present in the PLG-added samples, whereas all plasmids were completely degraded in the control samples.

[0081] Those skilled in the art readily recognize that the patented invention is well adapted to carry out the objects and obtain the objects and advantages mentioned and inherent. Growth hormone, ghrelin, analogs, plasmids, vectors, charged transfection-facilitating polypeptides, poly-L-glutamate, pharmaceutical compositions, therapies, electroporation methods, procedures, and other techniques described herein represent Several aspects of the invention are presented and are intended to be illustrative, not limiting in scope. For those skilled in the art, there will be some changes contained in the spirit of the art or defined by the pending claims.

Therefore, the present invention provides the method for host transfection therapeutic gene, and this method comprises by any aforementioned route of administration, or the replacement route known to those skilled in the art and suitable for specific use uses carrier of the present invention ( preferably as part of a composition). According to the present invention, the effective gene delivery of the vector to the host cell can be monitored by the therapeutic effect (such as the alleviation of certain symptoms associated with the particular disease being treated) or further by the delivered gene or the expression of the gene in the host cell other evidence to monitor (e.g., using polymerase chain reaction coupled to sequencing, northern blot or southern blot hybridization, or transcriptional analysis that detects nucleic acids in host cells, or using western blot analysis, antibody-mediated detection, mRNA or Protein half-life studies, or the use of specific assays to detect proteins or polypeptides encoded by delivered nucleic acids, or the effect of such delivery on protein or polypeptide levels or function).

[0083] The methods described here are by no means all-inclusive, and further methods suitable for a particular application will be apparent to those of ordinary skill. In addition, effective amounts of the compositions can be further approximated by analogy with compounds known to exert the desired effect.

[0084] Furthermore, actual dosages and schedules may vary depending upon whether the composition is used in combination with other pharmaceutical compositions, or upon intra-individual differences in pharmacokinetics, drug distribution and metabolism. Similarly, dosages for use in vitro will vary depending on the particular cell line used (eg, based on the number of vector acceptors present on the cell surface, or the replication capacity of the particular vector used to deliver the gene in the cell line). Furthermore, the amount of vector added to each cell will vary depending on the length and stability of the therapeutic gene inserted into the vector and the nature of the sequence, especially for parameters that need to be determined empirically, and may vary due to factors not determined by the methods of the present invention. inherent factors (such as costs associated with synthesis). Those skilled in the art can readily make any necessary adjustments according to the exigencies of a particular situation.

Sequence Listing

<110>Advisys

<120>

POLY-L increases in vivo delivery of nucleic acid constructs through poly-L-glutamic acid ("PLG")

<130>108328.00115-AVSI-0021P1

<140>10/395,709

<141>2003-03-24

<160>25

<170>PatentIn version 3.1

<210>1

<211>40

<212>PRT

<213> Artificial sequence

<220>

<223> Functional equivalents of GHRH

<400>1

His Val Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Ala Gln

1 5 10 15

Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Leu Asn Arg Gln Gln Gly

20 25 30

Glu Arg Asn Gln Glu Gln Gly Ala

35 40

<210>2

<211>40

<212>PRT

<213> Artificial sequence

<220>

<223> Functional equivalents of GHRH

<400>2

Tyr Ile Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Ala Gln

1 5 10 15

Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Leu Asn Arg Gln Gln Gly

20 25 30

Glu Arg Asn Gln Glu Gln Gly Ala

35 40

<210>3

<211>40

<212>PRT

<213> Artificial sequence

<220>

<223> Functional equivalents of GHRH

<400>3

Tyr Val Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Ala Gln

1 5 10 15

Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Leu Asn Arg Gln Gln Gly

20 25 30

Glu Arg Asn Gln Glu Gln Gly Ala

35 40

<210>4

<211>40

<212>PRT

<213> Artificial sequence

<220>

<223> Functional equivalents of GHRH

<400>4

Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Ala Gln

1 5 10 15

Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Leu Asn Arg Gln Gln Gly

20 25 30

Glu Arg Asn Gln Glu Gln Gly Ala

35 40

<210>5

<211>40

<212>PRT

<213> Artificial sequence

<220>

<223> This is the artificial sequence of (1-44)NH2

<400>5

Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln

1 5 10 15

Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly

20 25 30

Glu Arg Asn Gln Glu Gln Gly Ala

35 40

<210>6

<211>40

<212>PRT

<213> Artificial sequence

<220>

<223> This is the artificial sequence of GHRH(1-40)OH

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> Xaa at position 1 may be Tyr or His.

<220>

<221>MISC_FEATURE

<222>(2)..(2)

<223> Xaa at position 2 can be Ala, Val or Ile.

<220>

<221>MISC_FEATURE

<222>(15)..(15)

<223> Xaa at position 15 may be Ala, Val or Ile.

<220>

<221>MISC_FEATURE

<222>(27)..(27)

<223> Xaa at position 27 can be Met or Ile.

<220>

<221>MISC_FEATURE

<222>(28)..(28)

<223> Xaa at position 28 may be Ser or Asp.

<400>6

Xaa Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Xaa Gln

1 5 10 15

Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Xaa Xaa Arg Gln Gln Gly

20 25 30

Glu Arg Asn Gln Glu Gln Gly Ala

35 40

<210>7

<211>323

<212>DNA

<213> Artificial sequence

<220>

<223> This is the nucleic acid sequence of the eukaryotic promoter c5-12

<400>7

cggccgtccg ccctcggcac catcctcacg acacccaaat atggcgacgg gtgaggaatg 60

gtggggagtt atttttagag cggtgaggaa ggtgggcagg cagcaggtgt tggcgctcta 120

aaaataactc ccgggagtta tttttagagc ggaggaatgg tggacaccca aatatggcga 180

cggttcctca cccgtcgcca tatttgggtg tccgccctcg gccggggccg cattcctggg 240

ggccgggcgg tgctcccgcc cgcctcgata aaaggctccg gggccggcgg cggcccga 300

gctacccgga ggagcgggag gcg 323

<210>8

<211>190

<212>DNA

<213> Artificial sequence

<220>

<223> This is the nucleic acid sequence of the polyadenosine tail of human growth hormone (hGH)

<400>8

gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca 60

gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc 120

ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca 180

acctgtaggg 190

<210>9

<211>219

<212>DNA

<213> Artificial sequence

<220>

<223> This is the cDNA of porcine growth hormone releasing hormone

<400>9

atggtgctct gggtgttctt ctttgtgatc ctcaccctca gcaacagctc ccactgctcc 60

ccacctcccc ctttgaccct caggatgcgg cggcacgtag atgccatctt caccaacagc 120

taccggaagg tgctggccca gctgtccgcc cgcaagctgc tccaggacat cctgaacagg 180

cagcagggag agaggaacca agagcaagga gcataatga 219

<210>10

<211>40

<212>PRT

<213> Artificial sequence

<220>

<223> This is the amino acid sequence of porcine growth hormone releasing hormone

<400>10

Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln

1 5 10 15

Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly

20 25 30

Glu Arg Asn Gln Glu Gln Gly Ala

35 40

<210>11

<211>3534

<212>DNA

<213> Artificial sequence

<220>

<223> This is the nucleic acid sequence of the components operably linked to the HV-GHRH plasmid

<400>11

gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa ttggagctcc 60

accgcggtgg cggccgtccg ccctcggcac catcctcacg acacccaaat atggcgacgg 120

gtgaggaatg gtggggagtt atttttagag cggtgaggaa ggtgggcagg cagcaggtgt 180

tggcgctcta aaaataactc ccgggagtta tttttagagc ggaggaatgg tggacaccca 240

aatatggcga cggttcctca cccgtcgcca tatttgggtg tccgccctcg gccggggccg 300

cattcctggg ggccgggcgg tgctcccgcc cgcctcgata aaaggctccg gggccggcgg 360

cggcccacga gctacccgga ggagcgggag gcgccaagct ctagaactag tggatcccaa 420

ggcccaactc cccgaaccac tcagggtcct gtggacagct cacctagctg ccatggtgct 480

ctgggtgttc ttctttgtga tcctcaccct cagcaacagc tcccactgct ccccacctcc 540

ccctttgacc ctcaggatgc ggcggcacgt agatgccatc ttcaccaaca gctaccggaa 600

ggtgctggcc cagctgtccg cccgcaagct gctccaggac atcctgaaca ggcagcaggg 660

agagaggaac caagagcaag gagcataatg actgcaggaa ttcgatatca agcttatcgg 720

ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag 780

tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct 840

tctataatat tatggggtgg agggggggtgg tatggagcaa ggggcaagtt gggaagacaa 900

cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca caatcttggc 960

tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct cccgagttgt 1020

tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg tagagacggg 1080

gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc taccccacctt 1140

ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga 1200

ttttaaaata actataccag caggaggacg tccagacaca gcataggcta cctggccatg 1260

cccaaccggt gggacatttg agttgcttgc ttggcactgt cctctcatgc gttgggtcca 1320

ctcagtagat gcctgttgaa ttcgataccg tcgacctcga gggggggccc ggtaccagct 1380

tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca tagctgtttc 1440

ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 1500

gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 1560

ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 1620

ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 1680

cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 1740

cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 1800

accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 1860

acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 1920

cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 1980

acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 2040

atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 2100

agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 2160

acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 2220

gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 2280

gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 2340

gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 2400

gaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagaaga 2460

actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa 2520

gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca 2580

acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa 2640

agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat 2700

cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct 2760

gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc 2820

gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca 2880

gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca 2940

ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa 3000

cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct 3060

cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc 3120

cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt 3180

catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt 3240

caatcatgcg aaacgatcct catcctgtct cttgatcaga tcttgatccc ctgcgccatc 3300

agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca accttaccag 3360

agggcgcccc agctggcaat tccggttcgc ttgctgtcca taaaaccgcc cagtctagca 3420

actgttggga agggcgatcg gtgcggggcct cttcgctatt acgccagctg gcgaaagggg 3480

gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgac 3534

<210>12

<211>3534

<212>DNA

<213> Artificial sequence

<220>

<223> This is the nucleic acid sequence of the components operably linked to the TI-GHRH plasmid

<400>12

gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa ttggagctcc 60

accgcggtgg cggccgtccg ccctcggcac catcctcacg acacccaaat atggcgacgg 120

gtgaggaatg gtggggagtt atttttagag cggtgaggaa ggtgggcagg cagcaggtgt 180

tggcgctcta aaaataactc ccgggagtta tttttagagc ggaggaatgg tggacaccca 240

aatatggcga cggttcctca cccgtcgcca tatttgggtg tccgccctcg gccggggccg 300

cattcctggg ggccgggcgg tgctcccgcc cgcctcgata aaaggctccg gggccggcgg 360

cggcccacga gctacccgga ggagcgggag gcgccaagct ctagaactag tggatcccaa 420

ggcccaactc cccgaaccac tcagggtcct gtggacagct cacctagctg ccatggtgct 480

ctgggtgttc ttctttgtga tcctcaccct cagcaacagc tcccactgct ccccacctcc 540

ccctttgacc ctcaggatgc ggcggtatat cgatgccatc ttcaccaaca gctaccggaa 600

ggtgctggcc cagctgtccg cccgcaagct gctccaggac atcctgaaca ggcagcaggg 660

agagaggaac caagagcaag gagcataatg actgcaggaa ttcgatatca agcttatcgg 720

ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag 780

tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct 840

tctataatat tatggggtgg agggggggtgg tatggagcaa ggggcaagtt gggaagacaa 900

cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca caatcttggc 960

tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct cccgagttgt 1020

tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg tagagacggg 1080

gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc taccccacctt 1140

ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga 1200

ttttaaaata actataccag caggaggacg tccagacaca gcataggcta cctggccatg 1260

cccaaccggt gggacatttg agttgcttgc ttggcactgt cctctcatgc gttgggtcca 1320

ctcagtagat gcctgttgaa ttcgataccg tcgacctcga gggggggccc ggtaccagct 1380

tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca tagctgtttc 1440

ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 1500

gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 1560

ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 1620

ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 1680

cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 1740

cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 1800

accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 1860

acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 1920

cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 1980

acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 2040

atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 2100

agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 2160

acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 2220

gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 2280

gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 2340

gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 2400

gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagaaga 2460

actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa 2520

gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca 2580

acgctatgtc ctgatagcgg tccgccaacac ccagccggcc acagtcgatg aatccagaaa 2640

agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat 2700

cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct 2760

gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc 2820

gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca 2880

gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca 2940

ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa 3000

cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct 3060

cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc 3120

cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt 3180

catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt 3240

caatcatgcg aaacgatcct catcctgtct cttgatcaga tcttgatccc ctgcgccatc 3300

agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca accttaccag 3360

agggcgcccc agctggcaat tccggttcgc ttgctgtcca taaaaccgcc cagtctagca 3420

actgttggga agggcgatcg gtgcggggcct cttcgctatt acgccagctg gcgaaagggg 3480

gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgac 3534

<210>13

<211>3534

<212>DNA

<213> Artificial sequence

<220>

<223> This is the nucleic acid sequence of the components operably linked to the TV-GHRH plasmid

<400>13

gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa ttggagctcc 60

accgcggtgg cggccgtccg ccctcggcac catcctcacg acacccaaat atggcgacgg 120

gtgaggaatg gtggggagtt atttttagag cggtgaggaa ggtgggcagg cagcaggtgt 180

tggcgctcta aaaataactc ccgggagtta tttttagagc ggaggaatgg tggacaccca 240

aatatggcga cggttcctca cccgtcgcca tatttgggtg tccgccctcg gccggggccg 300

cattcctggg ggccgggcgg tgctcccgcc cgcctcgata aaaggctccg gggccggcgg 360

cggcccacga gctacccgga ggagcgggag gcgccaagct ctagaactag tggatcccaa 420

ggcccaactc cccgaaccac tcagggtcct gtggacagct cacctagctg ccatggtgct 480

ctgggtgttc ttctttgtga tcctcaccct cagcaacagc tcccactgct ccccacctcc 540

ccctttgacc ctcaggatgc ggcggtatgt agatgccatc ttcaccaaca gctaccggaa 600

ggtgctggcc cagctgtccg cccgcaagct gctccaggac atcctgaaca ggcagcaggg 660

agagaggaac caagagcaag gagcataatg actgcaggaa ttcgatatca agcttatcgg 720

ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag 780

tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct 840

tctataatat tatggggtgg agggggggtgg tatggagcaa ggggcaagtt gggaagacaa 900

cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca caatcttggc 960

tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct cccgagttgt 1020

tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg tagagacggg 1080

gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc taccccacctt 1140

ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga 1200

ttttaaaata actataccag caggaggacg tccagacaca gcataggcta cctggccatg 1260

cccaaccggt gggacatttg agttgcttgc ttggcactgt cctctcatgc gttgggtcca 1320

ctcagtagat gcctgttgaa ttcgataccg tcgacctcga gggggggccc ggtaccagct 1380

tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca tagctgtttc 1440

ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 1500

gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 1560

ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 1620

ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 1680

cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 1740

cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 1800

accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 1860

acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 1920

cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 1980

acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 2040

atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 2100

agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 2160

acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 2220

gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 2280

gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 2340

gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 2400

gaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagaaga 2460

actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa 2520

gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca 2580

acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa 2640

agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat 2700

cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct 2760

gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc 2820

gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca 2880

gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca 2940

ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa 3000

cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct 3060

cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc 3120

cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt 3180

catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt 3240

caatcatgcg aaacgatcct catcctgtct cttgatcaga tcttgatccc ctgcgccatc 3300

agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca accttaccag 3360

agggcgcccc agctggcaat tccggttcgc ttgctgtcca taaaaccgcc cagtctagca 3420

actgttggga agggcgatcg gtgcggggcct cttcgctatt acgccagctg gcgaaagggg 3480

gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgac 3534

<210>14

<211>3534

<212>DNA

<213> Artificial sequence

<220>

<223> This is the nucleic acid sequence of the components operably linked to the 15/27/28GHRH plasmid

<400>14

gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa ttggagctcc 60

accgcggtgg cggccgtccg ccctcggcac catcctcacg acacccaaat atggcgacgg 120

gtgaggaatg gtggggagtt atttttagag cggtgaggaa ggtgggcagg cagcaggtgt 180

tggcgctcta aaaataactc ccgggagtta tttttagagc ggaggaatgg tggacaccca 240

aatatggcga cggttcctca cccgtcgcca tatttgggtg tccgccctcg gccggggccg 300

cattcctggg ggccgggcgg tgctcccgcc cgcctcgata aaaggctccg gggccggcgg 360

cggcccacga gctacccgga ggagcgggag gcgccaagct ctagaactag tggatcccaa 420

ggcccaactc cccgaaccac tcagggtcct gtggacagct cacctagctg ccatggtgct 480

ctgggtgttc ttctttgtga tcctcaccct cagcaacagc tcccactgct ccccacctcc 540

ccctttgacc ctcaggatgc ggcggtatat cgatgccatc ttcaccaaca gctaccggaa 600

ggtgctggcc cagctgtccg cccgcaagct gctccaggac atcctgaaca ggcagcaggg 660

agagaggaac caagagcaag gagcataatg actgcaggaa ttcgatatca agcttatcgg 720

ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag 780

tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct 840

tctataatat tatggggtgg agggggggtgg tatggagcaa ggggcaagtt gggaagacaa 900

cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca caatcttggc 960

tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct cccgagttgt 1020

tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg tagagacggg 1080

gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc taccccacctt 1140

ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga 1200

ttttaaaata actataccag caggaggacg tccagacaca gcataggcta cctggccatg 1260

cccaaccggt gggacatttg agttgcttgc ttggcactgt cctctcatgc gttgggtcca 1320

ctcagtagat gcctgttgaa ttcgataccg tcgacctcga gggggggccc ggtaccagct 1380

tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca tagctgtttc 1440

ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 1500

gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 1560

ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 1620

ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 1680

cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 1740

cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 1800

accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 1860

acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 1920

cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 1980

acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 2040

atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 2100

agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 2160

acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 2220

gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 2280

gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 2340

gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 2400

gaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagaaga 2460

actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa 2520

gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca 2580

acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa 2640

agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat 2700

cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct 2760

gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc 2820

gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca 2880

gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagaaagg tgagatgaca 2940

ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa 3000

cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct 3060

cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc 3120

cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt 3180

catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt 3240

caatcatgcg aaacgatcct catcctgtct cttgatcaga tcttgatccc ctgcgccatc 3300

agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca accttaccag 3360

agggcgcccc agctggcaat tccggttcgc ttgctgtcca taaaaccgcc cagtctagca 3420

actgttggga agggcgatcg gtgcggggcct cttcgctatt acgccagctg gcgaaagggg 3480

gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgac 3534

<210>15

<211>3534

<212>DNA

<213> Artificial sequence

<220>

<223> This is the full plasmid sequence of wild-type GHRH

<400>15

gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa ttggagctcc 60

accgcggtgg cggccgtccg ccctcggcac catcctcacg acacccaaat atggcgacgg 120

gtgaggaatg gtggggagtt atttttagag cggtgaggaa ggtgggcagg cagcaggtgt 180

tggcgctcta aaaataactc ccgggagtta tttttagagc ggaggaatgg tggacaccca 240

aatatggcga cggttcctca cccgtcgcca tatttgggtg tccgccctcg gccggggccg 300

cattcctggg ggccgggcgg tgctcccgcc cgcctcgata aaaggctccg gggccggcgg 360

cggcccacga gctacccgga ggagcgggag gcgccaagct ctagaactag tggatcccaa 420

ggcccaactc cccgaaccac tcagggtcct gtggacagct cacctagctg ccatggtgct 480

ctgggtgttc ttctttgtga tcctcaccct cagcaacagc tcccactgct ccccacctcc 540

ccctttgacc ctcaggatgc ggcggtatgc agatgccatc ttcaccaaca gctaccggaa 600

ggtgctgggc cagctgtccg cccgcaagct gctccaggac atcatgagca ggcagcaggg 660

agagaggaac caagagcaag gagcataatg actgcaggaa ttcgatatca agcttatcgg 720

ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag 780

tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct 840

tctataatat tatggggtgg agggggggtgg tatggagcaa ggggcaagtt gggaagacaa 900

cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca caatcttggc 960

tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct cccgagttgt 1020

tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg tagagacggg 1080

gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc taccccacctt 1140

ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga 1200

ttttaaaata actataccag caggaggacg tccagacaca gcataggcta cctggccatg 1260

cccaaccggt gggacatttg agttgcttgc ttggcactgt cctctcatgc gttgggtcca 1320

ctcagtagat gcctgttgaa ttcgataccg tcgacctcga gggggggccc ggtaccagct 1380

tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca tagctgtttc 1440

ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 1500

gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 1560

ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 1620

ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 1680

cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 1740

cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 1800

accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 1860

acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 1920

cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 1980

acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 2040

atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 2100

agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 2160

acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 2220

gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 2280

gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 2340

gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 2400

gaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagaaga 2460

actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa 2520

gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca 2580

acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa 2640

agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat 2700

cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct 2760

gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc 2820

gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca 2880

gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca 2940

ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa 3000

cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct 3060

cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc 3120

cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt 3180

catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt 3240

caatcatgcg aaacgatcct catcctgtct cttgatcaga tcttgatccc ctgcgccatc 3300

agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca accttaccag 3360

agggcgcccc agctggcaat tccggttcgc ttgctgtcca taaaaccgcc cagtctagca 3420

actgttggga agggcgatcg gtgcggggcct cttcgctatt acgccagctg gcgaaagggg 3480

gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgac 3534

<210>16

<211>4260

<212>DNA

<213> Artificial sequence

<220>

<223> This is the sequence of the pSP-SEAP cDNA construct

<400>16

ggccgtccgc cttcggcacc atcctcacga cacccaaata tggcgacggg tgaggaatgg 60

tggggagtta tttttagagc ggtgaggaag gtgggcaggc agcaggtgtt ggcgctctaa 120

aaataactcc cgggagttat ttttagagcg gaggaatggt ggacacccaa atatggcgac 180

ggttcctcac ccgtcgccat atttgggtgt ccgccctcgg ccggggccgc attcctgggg 240

gccgggcggt gctcccgccc gcctcgataa aaggctccgg ggccggcggc ggcccacgag 300

ctacccggag gagcgggagg cgccaagctc tagaactagt ggatcccccg ggctgcagga 360

attcgatatc aagcttcgaa tcgcgaattc gcccaccatg ctgctgctgc tgctgctgct 420

gggcctgagg ctacagctct ccctgggcat catcccagtt gaggaggaga acccggactt 480

ctggaaccgc gaggcagccg aggccctggg tgccgccaag aagctgcagc ctgcacagac 540

agccgccaag aacctcatca tcttcctggg cgatgggatg ggggtgtcta cggtgacagc 600

tgccaggatc ctaaaagggc agaagaagga caaactgggg cctgagatac ccctggccat 660

ggaccgcttc ccatatgtgg ctctgtccaa gacatacaat gtagacaaac atgtgccaga 720

cagtggagcc acagccacgg cctacctgtg cggggtcaag ggcaacttcc agaccattgg 780

cttgagtgca gccgcccgct ttaaccagtg caacacgaca cgcggcaacg aggtcatctc 840

cgtgatgaat cgggccaaga aagcagggaa gtcagtggga gtggtaacca ccacacgagt 900

gcagcacgcc tcgccagccg gcacctacgc ccaacacggtg aaccgcaact ggtactcgga 960

cgccgacgtg cctgcctcgg cccgccagga ggggtgccag gacatcgcta cgcagctcat 1020

ctccaacatg gacattgacg tgatcctagg tggaggccga aagtacatgt ttcgcatggg 1080

aacccccagac cctgagtacc cagatgacta cagccaaggt gggaccaggc tggacgggaa 1140

gaatctggtg caggaatggc tggcgaagcg ccagggtgcc cggtatgtgt ggaaccgcac 1200

tgagctcatg caggcttccc tggacccgtc tgtgacccat ctcatgggtc tctttgagcc 1260

tggagacatg aaatacgaga tccaccgaga ctccaacactg gacccctccc tgatggagat 1320

gacagaggct gccctgcgcc tgctgagcag gaacccccgc ggcttcttcc tcttcgtgga 1380

gggtggtcgc atcgaccatg gtcatcatga aagcagggct taccgggcac tgactgagac 1440

gatcatgttc gacgacgcca ttgagagggc gggccagctc accagcgagg aggacacgct 1500

gagcctcgtc actgccgacc actcccacgt cttctccttc ggaggctacc ccctgcgagg 1560

gagctccatc ttcgggctgg cccctggcaa ggcccgggac aggaaggcct acacggtcct 1620

cctatacgga aacggtccag gctatgtgct caaggacggc gcccggccgg atgttaccga 1680

gagcgagagc gggagccccg agtatcggca gcagtcagca gtgcccctgg acgaagagac 1740

ccacgcaggc gaggacgtgg cggtgttcgc gcgcggcccg caggcgcacc tggttcacgg 1800

cgtgcaggag cagaccttca tagcgcacgt catggccttc gccgcctgcc tggagcccta 1860

caccgcctgc gacctggcgc cccccgccgg caccaccgac gccgcgcacc cgggttactc 1920

tagagtcggg gcggccggcc gcttcgagca gacatgataa gatacattga tgagtttgga 1980

caaaccacaa ctagaatgca gtgaaaaaaa tgctttatt gtgaaatttg tgatgctatt 2040

gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat 2100

tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac 2160

aaatgtggta aaatcgataa ggatccgtcg accgatgccc ttgagagcct tcaacccagt 2220

cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt 2280

tatcatgcaa ctcgtaggac aggtgccggc agcgctcttc cgcttcctcg ctcactgact 2340

cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 2400

ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 2460

aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 2520

acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 2580

gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 2640

ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 2700

gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 2760

cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 2820

taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 2880

atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga 2940

cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 3000

cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 3060

ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 3120

ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 3180

tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 3240

aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 3300

tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 3360

gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 3420

atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 3480

tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 3540

ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 3600

ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 3660

tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 3720

ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 3780

ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 3840

tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 3900

gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 3960

taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 4020

cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 4080

agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 4140

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 4200

ataaacaaat agggggttccg cgcacatttc cccgaaaagt gccacctgac gcgccctgta 4260

<210>17

<211>2710

<212>DNA

<213> Artificial sequence

<220>

<223> This is a plasmid vector with the ghrelin analog sequence encoded for mouse

sub-optimization

<400>17

tgtaatacga ctcactatag ggcgaattgg agctccaccg cggtggcggc cgtccgccct 60

cggcaccatc ctcacgacac ccaaatatgg cgacgggtga ggaatggtgg ggagttattt 120

ttagagcggt gaggaaggtg ggcaggcagc aggtgttggc gctctaaaaa taactcccgg 180

gagttattt tagagcggag gaatggtgga cacccaaata tggcgacggt tcctcacccg 240

tcgccatatt tgggtgtccg ccctcggccg gggccgcatt cctgggggcc gggcggtgct 300

cccgcccgcc tcgataaaag gctccggggc cggcggcggc ccacgagcta cccggaggag 360

cgggaggcgc caagcggatc ccaaggccca actccccgaa ccactcaggg tcctgtggac 420

agctcaccta gctgccatgg tgctctgggt gctctttgtg atcctcatcc tcaccagcgg 480

cagccactgc agcctgcctc ccagccctcc cttcaggatg cagaggcacg tggacgccat 540

cttcaccacc aactacagga agctgctgag ccagctgtac gccaggaagg tgatccagga 600

catcatgaac aagcagggcg agaggatcca ggagcagagg gccaggctga gctgataagc 660

ttatcggggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc 720

actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag 780

gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg 840

aagacaacct gtagggctcg aggggggcc cggtaccagc ttttgttccc tttagtgagg 900

gttaatttcg agcttggtct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 960

ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 1020

gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 1080

aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 1140

gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 1200

ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 1260

cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 1320

cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 1380

gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 1440

cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 1500

agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 1560

ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 1620

ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 1680

gatctcaaga agatcctttg atcttttcta cggggctagc gcttagaaga actcatccag 1740

cagacggtag aatgcaatac gttgagagtc tggagctgca ataccataca gaaccaggaa 1800

acggtcagcc cattcaccac ccagttcctc tgcaatgtca cgggtagcca gtgcaatgtc 1860

ctggtaacgg tctgcaacac ccagacgacc acagtcaatg aaaccagaga aacgaccatt 1920

ctcaaccatg atgttcggca ggcatgcatc accatgagta actaccaggt cctcaccatc 1980

cggcatacga gctttcagac gtgcaaacag ttcagccggt gccagaccct gatgttcctc 2040

atccaggtca tcctggtcaa ccagacctgc ttccatacgg gtacgagcac gttcaatacg 2100

atgttttgcc tggtggtcaa acggacaggt agctgggtcc agggtgtgca gacgacgcat 2160

tgcatcagcc atgatagaaa ctttctctgc cggagccagg tgagaagaca gcaggtcctg 2220

acccggaact tcacccagca gcagccagtc acgaccagct tcagtaacta catccagaac 2280

tgcagcacac ggaacaccag tggttgccag ccaagacaga cgagctgctt catcctgcag 2340

ttcattcaga gcaccagaca ggtcagtttt aacaaacaga actggacgac cctgtgcaga 2400

cagacggaaa acagctgcat cagagcaacc aatggtctgc tgtgcccagt cataaccaaa 2460

cagacgttca acccaggctg ccggagaacc tgcatgcaga ccatcctgtt caatcatgcg 2520

aaacgatcct catcctgtct cttgatcaga tcttgatccc ctgcgccatc agatccttgg 2580

cggcaagaaa gccatccagt ttactttgca gggcttccca accttaccag agggcgcccc 2640

agctggcaat tccggttcgc ttgctgtcca taaaaccgcc cagtctagca actgttggga 2700

agggcgatcg 2710

<210>18

<211>2713

<212>DNA

<213> Artificial sequence

<220>

<223> This is a plasmid vector with the ghrelin analog sequence encoded for rat

sub-optimization

<400>18

tgtaatacga ctcactatag ggcgaattgg agctccaccg cggtggcggc cgtccgccct 60

cggcaccatc ctcacgacac ccaaatatgg cgacgggtga ggaatggtgg ggagttattt 120

ttagagcggt gaggaaggtg ggcaggcagc aggtgttggc gctctaaaaa taactcccgg 180

gagttattt tagagcggag gaatggtgga cacccaaata tggcgacggt tcctcacccg 240

tcgccatatt tgggtgtccg ccctcggccg gggccgcatt cctgggggcc gggcggtgct 300

cccgcccgcc tcgataaaag gctccggggc cggcggcggc ccacgagcta cccggaggag 360

cgggaggcgc caagcggatc ccaaggccca actccccgaa ccactcaggg tcctgtggac 420

agctcaccta gctgccatgg ccctgtgggt gttcttcgtg ctgctgaccc tgaccagcgg 480

aagccactgc agcctgcctc ccagccctcc cttcagggtg cgccggcacg ccgacgccat 540

cttcaccagc agctacagga ggatcctggg ccagctgtac gctaggaagc tcctgcacga 600

gatcatgaac aggcagcagg gcgagaggaa ccaggagcag aggagcaggt tcaactgata 660

agcttatcgg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt 720

gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac 780

taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt 840

gggaagacaa cctgtagggc tcgagggggg gcccggtacc agcttttgtt ccctttagtg 900

agggttaatt tcgagcttgg tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 960

ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 1020

caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 1080

aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 1140

atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 1200

cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 1260

ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 1320

gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaaccccccc gttcagcccg 1380

accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 1440

cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 1500

cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 1560

gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 1620

aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 1680

aaggatctca agaagatcct ttgatctttt ctacggggct agcgcttaga agaactcatc 1740

cagcagacgg tagaatgcaa tacgttgaga gtctggagct gcaataccat acagaaccag 1800

gaaacggtca gcccattcac cacccagttc ctctgcaatg tcacgggtag ccagtgcaat 1860

gtcctggtaa cggtctgcaa cacccagacg accacagtca atgaaaccag agaaacgacc 1920

attctcaacc atgatgttcg gcaggcatgc atcaccatga gtaactacca ggtcctcacc 1980

atccggcata cgagctttca gacgtgcaaa cagttcagcc ggtgccagac cctgatgttc 2040

ctcatccagg tcatcctggt caaccagacc tgcttccata cgggtacgag cacgttcaat 2100

acgatgtttt gcctggtggt caaacggaca ggtagctggg tccagggtgt gcagacgacg 2160

cattgcatca gccatgatag aaactttctc tgccggagcc aggtgagaag acagcaggtc 2220

ctgacccgga acttcaccca gcagcagcca gtcacgacca gcttcagtaa ctacatccag 2280

aactgcagca cacggaacac cagtggttgc cagccaagac agacgagctg cttcatcctg 2340

cagttcattc agagcaccag acaggtcagt tttaacaaac agaactggac gaccctgtgc 2400

agacagacgg aaaacagctg catcagagca accaatggtc tgctgtgccc agtcataacc 2460

aaacagacgt tcaacccagg ctgccggaga acctgcatgc agaccatcct gttcaatcat 2520

gcgaaacgat cctcatcctg tctcttgatc agatcttgat cccctgcgcc atcagatcct 2580

tggcggcaag aaagccatcc agtttacttt gcagggcttc ccaaccttac cagagggcgc 2640

cccagctggc aattccggtt cgcttgctgt ccataaaacc gcccagtcta gcaactgttg 2700

ggaagggcga tcg 2713

<210>19

<211>2704

<212> DNA

<213> Artificial sequence

<220>

<223> This is a plasmid vector with the ghrelin analogue sequence modified with codons for bovine

optimization

<400>19

tgtaatacga ctcactatag ggcgaattgg agctccaccg cggtggcggc cgtccgccct 60

cggcaccatc ctcacgacac ccaaatatgg cgacgggtga ggaatggtgg ggagttattt 120

ttagagcggt gaggaaggtg ggcaggcagc aggtgttggc gctctaaaaa taactcccgg 180

gagttattt tagagcggag gaatggtgga cacccaaata tggcgacggt tcctcacccg 240

tcgccatatt tgggtgtccg ccctcggccg gggccgcatt cctgggggcc gggcggtgct 300

cccgcccgcc tcgataaaag gctccggggc cggcggcggc ccacgagcta cccggaggag 360

cgggaggcgc caagcggatc ccaaggccca actccccgaa ccactcaggg tcctgtggac 420

agctcaccta gctgccatgg tgctgtgggt gttcttcctg gtgaccctga ccctgagcag 480

cggctcccac ggctccctgc cctcccagcc tctgcgcatc cctcgctacg ccgacgccat 540

cttcaccaac agctaccgca aggtgctcgg ccagctcagc gcccgcaagc tcctgcagga 600

catcatgaac cggcagcagg gcgagcgcaa ccaggagcag ggagcctgat aagcttatcg 660

gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca 720

gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc 780

ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca 840

acctgtaggg ctcgagggggg ggcccggtac cagcttttgt tccctttagt gagggttaat 900

ttcgagcttg gtcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 960

ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 1020

acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 1080

cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 1140

caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 1200

gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 1260

tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 1320

aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 1380

ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 1440

cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 1500

tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc 1560

tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 1620

ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 1680

aagaagatcc tttgatcttt tctacggggc tagcgcttag aagaactcat ccagcagacg 1740

gtagaatgca atacgttgag agtctggagc tgcaatacca tacagaacca ggaaacggtc 1800

agcccattca ccaccagtt cctctgcaat gtcacgggta gccagtgcaa tgtcctggta 1860

acggtctgca acacccagac gaccacagtc aatgaaacca gagaaacgac cattctcaac 1920

catgatgttc ggcaggcatg catcaccatg agtaactacc aggtcctcac catccggcat 1980

acgagctttc agacgtgcaa acagttcagc cggtgccaga ccctgatgtt cctcatccag 2040

gtcatcctgg tcaaccagac ctgcttccat acgggtacga gcacgttcaa tacgatgttt 2100

tgcctggtgg tcaaacggac aggtagctgg gtccagggtg tgcagacgac gcattgcatc 2160

agccatgata gaaactttct ctgccggagc caggtgagaa gacagcaggt cctgacccgg 2220

aacttcaccc agcagcagcc agtcacgacc agcttcagta actacatcca gaactgcagc 2280

acacggaaca ccagtggttg ccagccaaga cagacgagct gcttcatcct gcagttcatt 2340

cagagcacca gacagtcag ttttaacaaa cagaactgga cgaccctgtg cagacagacg 2400

gaaaacagct gcatcagagc aaccaatggt ctgctgtgcc cagtcataac caaacagacg 2460

ttcaacccag gctgccggag aacctgcatg cagaccatcc tgttcaatca tgcgaaacga 2520

tcctcatcct gtctcttgat cagatcttga tcccctgcgc catcagatcc ttggcggcaa 2580

gaaagccatc cagtttactt tgcagggctt cccaacctta ccagagggcg caccagctgg 2640

caattccggt tcgcttgctg tccataaaac cgcccagtct agcaactgtt gggaagggcg 2700

atcg 2704

<210>20

<211>2704

<212>DNA

<213> Artificial sequence

<220>

<223> This is a plasmid vector with a ghrelin analogue sequence that was directed against

Codon optimization for sheep

<400>20

tgtaatacga ctcactatag ggcgaattgg agctccaccg cggtggcggc cgtccgccct 60

cggcaccatc ctcacgacac ccaaatatgg cgacgggtga ggaatggtgg ggagttatt 120

ttagagcggt gaggaaggtg ggcaggcagc aggtgttggc gctctaaaaa taactcccgg 180

gagttattt tagagcggag gaatggtgga cacccaaata tggcgacggt tcctcacccg 240

tcgccatatt tgggtgtccg ccctcggccg gggccgcatt cctgggggcc gggcggtgct 300

cccgcccgcc tcgataaaag gctccggggc cggcggcggc ccacgagcta cccggaggag 360

cgggaggcgc caagcggatc ccaaggccca actccccgaa ccactcaggg tcctgtggac 420

agctcaccta gctgccatgg tgctgtgggt gttcttcctg gtgaccctga ccctgagcag 480

cggaagccac ggcagcctgc ccagccagcc cctgaggatc cctaggtacg ccgacgccat 540

cttcaccaac agctacagga agatcctggg ccagctgagc gctaggaagc tcctgcagga 600

catcatgaac aggcagcagg gcgagaggaa ccaggagcag ggcgcctgat aagcttatcg 660

gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca 720

gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc 780

ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca 840

acctgtaggg ctcgagggggg ggcccggtac cagcttttgt tccctttagt gagggttaat 900

ttcgagcttg gtcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 960

ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 1020

acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 1080

cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 1140

caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 1200

gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 1260

tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 1320

aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 1380

ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 1440

cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 1500

tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc 1560

tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 1620

ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 1680

aagaagatcc tttgatcttt tctacggggc tagcgcttag aagaactcat ccagcagacg 1740

gtagaatgca atacgttgag agtctggagc tgcaatacca tacagaacca ggaaacggtc 1800

agcccattca ccaccagtt cctctgcaat gtcacgggta gccagtgcaa tgtcctggta 1860

acggtctgca acacccagac gaccacagtc aatgaaacca gagaaacgac cattctcaac 1920

catgatgttc ggcaggcatg catcaccatg agtaactacc aggtcctcac catccggcat 1980

acgagctttc agacgtgcaa acagttcagc cggtgccaga ccctgatgtt cctcatccag 2040

gtcatcctgg tcaaccagac ctgcttccat acgggtacga gcacgttcaa tacgatgttt 2100

tgcctggtgg tcaaacggac aggtagctgg gtccagggtg tgcagacgac gcattgcatc 2160

agccatgata gaaactttct ctgccggagc caggtgagaa gacagcaggt cctgacccgg 2220

aacttcaccc agcagcagcc agtcacgacc agcttcagta actacatcca gaactgcagc 2280

acacggaaca ccagtggttg ccagccaaga cagacgagct gcttcatcct gcagttcatt 2340

cagagcacca gacagtcag ttttaacaaa cagaactgga cgaccctgtg cagacagacg 2400

gaaaacagct gcatcagagc aaccaatggt ctgctgtgcc cagtcataac caaacagacg 2460

ttcaacccag gctgccggag aacctgcatg cagaccatcc tgttcaatca tgcgaaacga 2520

tcctcatcct gtctcttgat cagatcttga tcccctgcgc catcagatcc ttggcggcaa 2580

gaaagccatc cagtttactt tgcagggctt cccaacctta ccagagggcg ccccagctgg 2640

caattccggt tcgcttgctg tccataaaac cgcccagtct agcaactgtt gggaagggcg 2700

atcg 2704

<210>21

<211>2713

<212>DNA

<213> Artificial sequence

<220>

<223> This is a plasmid vector with the ghrelin analogue sequence modified with codons for chicken

optimization

<400>21

tgtaatacga ctcactatag ggcgaattgg agctccaccg cggtggcggc cgtccgccct 60

cggcaccatc ctcacgacac ccaaatatgg cgacgggtga ggaatggtgg ggagttatt 120

ttagagcggt gaggaaggtg ggcaggcagc aggtgttggc gctctaaaaa taactcccgg 180

gagttattt tagagcggag gaatggtgga cacccaaata tggcgacggt tcctcacccg 240

tcgccatatt tgggtgtccg ccctcggccg gggccgcatt cctgggggcc gggcggtgct 300

cccgcccgcc tcgataaaag gctccggggc cggcggcggc ccacgagcta cccggaggag 360

cgggaggcgc caagcggatc ccaaggccca actccccgaa ccactcaggg tcctgtggac 420

agctcaccta gctgccatgg ccctgtgggt gttctttgtg ctgctgaccc tgacctccgg 480

aagccactgc agcctgccac ccagcccacc cttccgcgtc aggcgccacg ccgacggcat 540

cttcagcaag gcctaccgca agctcctggg ccagctgagc gcacgcaact acctgcacag 600

cctgatggcc aagcgcgtgg gcagcggact gggagacgag gccgagcccc tgagctgata 660

agcttatcgg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt 720

gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac 780

taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt 840

gggaagacaa cctgtagggc tcgagggggg gcccggtacc agcttttgtt ccctttagtg 900

agggttaatt tcgagcttgg tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 960

ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 1020

caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 1080

aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 1140

atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 1200

cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 1260

ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 1320

gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaaccccccc gttcagcccg 1380

accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 1440

cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 1500

cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 1560

gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 1620

aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 1680

aaggatctca agaagatcct ttgatctttt ctacggggct agcgcttaga agaactcatc 1740

cagcagacgg tagaatgcaa tacgttgaga gtctggagct gcaataccat acagaaccag 1800

gaaacggtca gcccattcac cacccagttc ctctgcaatg tcacgggtag ccagtgcaat 1860

gtcctggtaa cggtctgcaa cacccagacg accacagtca atgaaaccag agaaacgacc 1920

attctcaacc atgatgttcg gcaggcatgc atcaccatga gtaactacca ggtcctcacc 1980

atccggcata cgagctttca gacgtgcaaa cagttcagcc ggtgccagac cctgatgttc 2040

ctcatccagg tcatcctggt caaccagacc tgcttccata cgggtacgag cacgttcaat 2100

acgatgtttt gcctggtggt caaacggaca ggtagctggg tccagggtgt gcagacgacg 2160

cattgcatca gccatgatag aaactttctc tgccggagcc aggtgagaag acagcaggtc 2220

ctgacccgga acttcaccca gcagcagcca gtcacgacca gcttcagtaa ctacatccag 2280

aactgcagca cacggaacac cagtggttgc cagccaagac agacgagctg cttcatcctg 2340

cagttcattc agagcaccag acaggtcagt tttaacaaac agaactggac gaccctgtgc 2400

agacagacgg aaaacagctg catcagagca accaatggtc tgctgtgccc agtcataacc 2460

aaacagacgt tcaacccagg ctgccggaga acctgcatgc agaccatcct gttcaatcat 2520

gcgaaacgat cctcatcctg tctcttgatc agatcttgat cccctgcgcc atcagatcct 2580

tggcggcaag aaagccatcc agtttacttt gcagggcttc ccaaccttac cagagggcgc 2640

cccagctggc aattccggtt cgcttgctgt ccataaaacc gcccagtcta gcaactgttg 2700

ggaagggcga tcg 2713

<210>22

<211>55

<212>DNA

<213> Artificial sequence

<220>

<223> This is the sequence of the 5' untranslated region of human growth hormone

<400>22

caaggcccaa ctccccgaac cactcagggt cctgtggaca gctcacctag ctgcc 55

<210>23

<211>782

<212>DNA

<213> Artificial sequence

<220>

<223> This is the nucleic acid sequence of the origin of replication of plasmid pUC-18

<400>23

tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 60

tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 120

aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 180

tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 240

tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 300

cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 360

agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 420

tccaagctgg gctgtgtgca cgaaccccccc gttcagcccg accgctgcgc cttatccggt 480

aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 540

ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 600

cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 660

accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 720

ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 780

tt 782

<210>24

<211>5

<212>DNA

<213> Artificial sequence

<220>

<223> This is the NEO ribosome binding site

<400>24

tcctc 5

<210>25

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> This is the nucleic acid sequence of the prokaryotic PNEO promoter

<400>25

accttaccag agggcgcccc agctggcaa 29

Claims (90)

1. A composition comprising: (a) a nucleic acid expression construct; and (b) a charged transfection-promoting polypeptide associated therewith; Wherein the molar ratio of the charged transfection-promoting polypeptide to the nucleic acid expression construct includes 1 to 5000 moles of the charged transfection-promoting polypeptide per mole of the nucleic acid expression construct. 2. The composition of claim 1, wherein the charged transfection-facilitating polypeptide comprises poly-L-glutamic acid. 3. The composition of claim 1, wherein the molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct is equal to 1200 moles or less of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 4. The composition of claim 1, wherein the molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct is equal to 1 mole of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 5. The composition of claim 1, wherein the nucleic acid expression vector has an average molecular length of about 2000 to about 5000 nucleotide base pairs. 6. The composition of claim 1, wherein the charged transfection-facilitating polypeptide has an average molecular weight of about 400 to about 30,000 Da. 7. The composition of claim 1, wherein the nucleic acid expression vector has an average molecular length of about 5000 nucleotide base pairs, and the charged transfection-facilitating polypeptide has an average molecular weight of about 10900 Da. 8. The composition of claim 1, wherein the nucleic acid expression construct comprises SeqID#11, SeqID#12, SeqID#13, SeqID#14, SeqID#17, SeqID#18, SeqID#19, SeqID#20, or SeqID# twenty one. 9. The composition of claim 1, wherein the nucleic acid expression construct comprises a gene encoding growth hormone releasing hormone ("GHRH") or a biologically functional equivalent thereof. 10. The composition of claim 9, wherein the encoded GHRH is a biologically active polypeptide, and the encoded GHRH biologically functional equivalent is a polypeptide that is engineered to have a different amino acid sequence but simultaneously has similar or improved biological activity when compared with the GHRH polypeptide . 11. The composition of claim 9, wherein the encoded GHRH or its biologically functional equivalent has the following general formula (SEQ ID #6): -X ₁ -X ₂ -DAIFTNSYRKVL-X ₃ -QLSARKLLQDI-X ₄ -X ₅ -RQQGERNQEQGA-OH Wherein the general formula has the following characteristics: _X is the D- or L-isomer of tyrosine ("Y") or histidine ("H"); _X is the D- or L-isomer of alanine ("A"), valine ("V") or isoleucine ("I"); _X is the D- or L-isomer of alanine ("A") or glycine ("G"); _X is the D- or L-isomer of methionine ("M") or leucine ("L"); _X5 is the D- or L-isomer of serine ("S") or asparagine ("N"); or a combination thereof. 12. The composition of claim 1, wherein the nucleic acid expression construct encodes a polypeptide sequence comprising SeqID#1, SeqID#2, SeqID#3, SeqID#4 or SeqID#5. 13. Compositions comprising: (a) a nucleic acid expression construct; and (b) a poly-L-glutamic acid polypeptide associated therewith; Wherein the molar ratio of the charged transfection-promoting polypeptide to the nucleic acid expression construct includes 1 to 5000 moles of the charged transfection-promoting polypeptide per mole of the nucleic acid expression construct. 14. The composition of claim 13, wherein the molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct is equal to 1200 moles or less of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 15. The composition of claim 13, wherein the molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct is equal to 1 mole of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 16. The composition of claim 13, wherein the nucleic acid expression vector has an average molecular length of about 2000 to about 5000 nucleotide base pairs. 17. The composition of claim 13, wherein the charged transfection-facilitating polypeptide has an average molecular weight of about 400 to about 30,000 Da. 18. The composition of claim 13, wherein the nucleic acid expression vector has an average molecular length of about 5000 nucleotide base pairs, and the charged transfection-facilitating polypeptide has an average molecular weight of about 10900 Da. 19. The composition of claim 13, wherein the nucleic acid expression construct comprises SeqID#11, SeqID#12, SeqID#13, SeqID#14, SeqID#17, SeqID#18, SeqID#19, SeqID#20, or SeqID# twenty one. 20. The composition of claim 13, wherein the nucleic acid expression construct comprises a gene encoding growth hormone releasing hormone ("GHRH") or a biologically functional equivalent thereof. 21. The composition of claim 20, wherein the encoded GHRH is a biologically active polypeptide, and the encoded GHRH biologically functional equivalent is a polypeptide that has been engineered to have a different amino acid sequence but simultaneously has similar or improved biological activity when compared with the GHRH polypeptide . 22. The composition of claim 20, wherein the encoded GHRH or its biologically functional equivalent has the general formula (SEQ ID #6): -X ₁ -X ₂ -DAIFTNSYRKVL-X ₃ -QLSARKLLQDI-X ₄ -X ₅ -RQQGERNQEQGA-OH Wherein the general formula has the following characteristics: _X is the D- or L-isomer of tyrosine ("Y") or histidine ("H"); _X is the D- or L-isomer of alanine ("A"), valine ("V") or isoleucine ("I"); _X is the D- or L-isomer of alanine ("A") or glycine ("G"); _X is the D- or L-isomer of methionine ("M") or leucine ("L"); _X5 is the D- or L-isomer of serine ("S") or asparagine ("N"); or a combination thereof. 23. The composition of claim 13, wherein the nucleic acid expression construct encodes a polypeptide sequence comprising SeqID #1, SeqID #2, SeqID #3, SeqID #4 or SeqID #5. 24. Compositions comprising: (a) a nucleic acid expression construct encoding growth hormone releasing hormone ("GHRH") or a functional equivalent thereof; and (b) a poly-L-glutamic acid polypeptide associated therewith; Wherein the molar ratio of the charged transfection-promoting polypeptide to the nucleic acid expression construct includes 1 to 5000 moles of the charged transfection-promoting polypeptide per mole of the nucleic acid expression construct. 25. The composition of claim 24, wherein the molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct is equal to 1200 moles or less of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 26. The composition of claim 24, wherein the molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct is equal to 1 mole of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 27. The composition of claim 24, wherein the nucleic acid expression vector has an average molecular length of about 2000 to about 5000 nucleotide base pairs. 28. The composition of claim 24, wherein the charged transfection-facilitating polypeptide has an average molecular weight of about 400 to about 30,000 Da. 29. The composition of claim 24, wherein the nucleic acid expression vector has an average molecular length of about 5000 nucleotide base pairs, and the charged transfection-facilitating polypeptide has an average molecular weight of about 10900 Da. 30. The composition of claim 24, wherein the nucleic acid expression construct comprises SeqID#11, SeqID#12, SeqID#13, SeqID#14, SeqID#17, SeqID#18, SeqID#19, SeqID#20, or SeqID# twenty one. 31. The composition of claim 24, wherein the encoded GHRH is a biologically active polypeptide, and the encoded GHRH biologically functional equivalent is engineered to have a different amino acid sequence but simultaneously have similar or improved biological activity when compared with the GHRH polypeptide peptide. 32. The composition of claim 24, wherein the encoded GHRH or its biologically functional equivalent has the general formula (SEQ ID #6): -X ₁ -X ₂ -DAIFTNSYRKVL-X ₃ -QLSARKLLQDI-X ₄ -X ₅ -RQQGERNQEQGA-OH Wherein the general formula has the following characteristics: _X is the D- or L-isomer of tyrosine ("Y") or histidine ("H"); _X is the D- or L-isomer of alanine ("A"), valine ("V") or isoleucine ("I"); _X is the D- or L-isomer of alanine ("A") or glycine ("G"); _X is the D- or L-isomer of methionine ("M") or leucine ("L"); _X5 is the D- or L-isomer of serine ("S") or asparagine ("N"); or a combination thereof. 33. The composition of claim 24, wherein the nucleic acid expression construct encodes a polypeptide sequence comprising SeqID #1, SeqID #2, SeqID #3, SeqID #4 or SeqID #5. 34. The composition comprising: (a) a nucleic acid expression construct encoding growth hormone releasing hormone ("GHRH") or a functional equivalent thereof; and (b) a charged transfection-promoting polypeptide associated therewith; Wherein the molar ratio of the charged transfection-promoting polypeptide to the nucleic acid expression construct includes 1 to 5000 moles of the charged transfection-promoting polypeptide per mole of the nucleic acid expression construct. 35. The composition of claim 34, wherein the molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct is equal to 1200 moles or less of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 36. The composition of claim 34, wherein the molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct is equal to 1 mole of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 37. The composition of claim 34, wherein the nucleic acid expression vector has an average molecular length of about 2000 to about 5000 nucleotide base pairs. 38. The composition of claim 34, wherein the charged transfection-facilitating polypeptide has an average molecular weight of about 400 to about 30,000 Da. 39. The composition of claim 34, wherein the nucleic acid expression vector has an average molecular length of about 5000 nucleotide base pairs, and the charged transfection-facilitating polypeptide has an average molecular weight of about 10900 Da. 40. The composition of claim 34, wherein the charged transfection-facilitating polypeptide comprises poly-L-glutamic acid. 41. The composition of claim 34, wherein the nucleic acid expression construct comprises SeqID#11, SeqID#12, SeqID#13, SeqID#14, SeqID#17, SeqID#18, SeqID#19, SeqID#20, or SeqID# twenty one. 42. The composition of claim 34, wherein the encoded GHRH is a biologically active polypeptide, and the encoded GHRH biologically functional equivalent is engineered to have a different amino acid sequence but simultaneously have similar or improved biological activity when compared with the GHRH polypeptide peptide. 43. The composition of claim 34, wherein the encoded GHRH or its biologically functional equivalent has the general formula (SEQ ID #6): -X ₁ -X ₂ -DAIFTNSYRKVL-X ₃ -QLSARKLLQDI-X ₄ -X ₅ -RQQGERNQEQGA-OH Wherein the general formula has the following characteristics: _X is the D- or L-isomer of tyrosine ("Y") or histidine ("H"); _X is the D- or L-isomer of alanine ("A"), valine ("V") or isoleucine ("I"); _X is the D- or L-isomer of alanine ("A") or glycine ("G"); _X is the D- or L-isomer of methionine ("M") or leucine ("L"); _X5 is the D- or L-isomer of serine ("S") or asparagine ("N"); or a combination thereof. 44. The composition of claim 34, wherein the nucleic acid expression construct encodes a polypeptide sequence comprising SeqID #1, SeqID #2, SeqID #3, SeqID #4 or SeqID #5. 45. A method of introducing a nucleic acid expression construct into cells of a tissue selected by a recipient, comprising: (a) placing a plurality of electrodes into the selected tissue, wherein the plurality of electrodes are arranged in a certain spatial relationship; (b) introducing a nucleic acid expression construct associated with a charged transfection-facilitating polypeptide; and (c) applying constant current electric pulses to a plurality of electrodes; Wherein the molar ratio of the charged transfection-promoting polypeptide to the nucleic acid expression construct includes from 1 to 5000 moles of the charged transfection-promoting polypeptide per mole of the nucleic acid expression construct. 46. The composition of claim 45, wherein the nucleic acid expression construct comprises SeqED#11, SeqID#12, SeqID#13, SeqID#14, SeqID#17, SeqID#18, SeqID#19, SeqID#20 or SeqID#21 . 47. The method of claim 45, wherein the cells of the selected tissue comprise somatic cells, stem cells, or germ cells. 48. The method of claim 45, wherein the selected tissue of the recipient comprises muscle. 49. The method of claim 45, wherein the charged transfection-facilitating polypeptide comprises poly-L-glutamate. 50. The method of claim 45, wherein the molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct is equal to 1200 moles or less of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 51. The method of claim 45, wherein the molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct is equal to 1 mole of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 52. The method of claim 45, wherein the plurality of electrodes are constructed of a material capable of making galvanic contact with the tissue. 53. The method of claim 45, wherein the nucleic acid expression construct comprises a gene encoding growth hormone releasing hormone ("GHRH") or a biologically functional equivalent thereof. 54. The method of claim 53, wherein the encoded GHRH or a biologically functional equivalent thereof is expressed in recipient tissue-specific cells. 55. The method of claim 53, wherein the encoded GHRH is a biologically active polypeptide, and the encoded GHRH biologically functional equivalent is a polypeptide that has been engineered to have a different amino acid sequence when compared with the GHRH polypeptide but at the same time has similar or improved biological activity . 56. The method of claim 53, wherein the encoded GHRH or its biologically functional equivalent has the general formula (SEQ ID #6): -X ₁ -X ₂ -DAIFTNSYRKVL-X ₃ -QLSARKLLQDI-X ₄ -X ₅ -RQQGERNQEQGA-OH Wherein the general formula has the following characteristics: _X is the D- or L-isomer of tyrosine ("Y") or histidine ("H"); _X is the D- or L-isomer of alanine ("A"), valine ("V") or isoleucine ("I"); _X is the D- or L-isomer of alanine ("A") or glycine ("G"); _X is the D- or L-isomer of methionine ("M") or leucine ("L"); _X5 is the D- or L-isomer of serine ("S") or asparagine ("N"); or a combination thereof. 57. The method of claim 45, wherein the nucleic acid expression construct encodes a polypeptide sequence comprising SeqID #1, SeqID #2, SeqID #3, SeqID #4 or SeqID #5. 58. A method of introducing a nucleic acid expression construct into a muscle cell of the body, comprising: (a) placing a plurality of electrodes into the selected tissue, wherein the plurality of electrodes are arranged in a certain spatial relationship; (b) introducing a nucleic acid expression construct associated with a charged transfection-promoting polypeptide; wherein the charged transfection-promoting polypeptide comprises a poly-L-glutamic acid polypeptide; (c) applying electrical pulses to a plurality of electrodes; wherein the nucleic acid expression construct encodes growth hormone releasing hormone ("GHRH") or a biologically functional equivalent thereof; and The molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct comprises from 1 to 5000 moles of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 59. The method of claim 58, wherein the nucleic acid expression vector has an average molecular length of about 2000 to about 5000 nucleotide base pairs. 60. The method of claim 58, wherein the charged transfection-facilitating polypeptide has an average molecular weight of about 400 to about 30,000 Da. 61. The method of claim 58, wherein the nucleic acid expression vector has an average molecular length of about 5000 nucleotide base pairs and the charged transfection-facilitating polypeptide has an average molecular weight of about 10900 Da. 62. The method of claim 58, wherein the nucleic acid expression construct comprises SeqED#11, SeqID#12, SeqID#13, SeqID#14, SeqID#17, SeqID#18, SeqID#19, SeqID#20, or SeqID#21. 63. The method of claim 58, wherein the molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct is equal to 1200 moles or less of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 64. The method of claim 58, wherein the molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct is equal to 1 mole of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 65. The method of claim 58, wherein the plurality of electrodes are constructed of a material capable of making galvanic contact with the tissue. 66. The method of claim 58, wherein introduction of the nucleic acid expression construct into the recipient's muscle cells elicits expression of the encoded growth hormone releasing hormone ("GHRH") or a biologically functional equivalent thereof. 67. The method of claim 58, wherein the encoded GHRH or a biologically functional equivalent thereof is expressed in tissue-specific cells of the recipient. 68. The method of claim 58, wherein the encoded GHRH is a biologically active polypeptide, and the encoded GHRH biologically functional equivalent is a polypeptide that has been engineered to have a different amino acid sequence when compared with the GHRH polypeptide but at the same time has similar or improved biological activity . 69. The method of claim 58, wherein the encoded GHRH or its biologically functional equivalent has the general formula (SEQ ID #6): -X ₁ -X ₂ -DAIFTNSYRKVL-X ₃ -QLSARKLLQDI-X ₄ -X ₅ -RQQGERNQEQGA-OH Wherein the general formula has the following characteristics: _X is the D- or L-isomer of tyrosine ("Y") or histidine ("H"); _X is the D- or L-isomer of alanine ("A"), valine ("V") or isoleucine ("I"); _X is the D- or L-isomer of alanine ("A") or glycine ("G"); _X is the D- or L-isomer of methionine ("M") or leucine ("L"); _X5 is the D- or L-isomer of serine ("S") or asparagine ("N"); or a combination thereof. 70. The method of claim 58, wherein the nucleic acid expression construct encodes a polypeptide sequence comprising SeqID #1, SeqID #2, SeqID #3, SeqID #4, or SeqID #5. 71. A method for increasing the stability of a nucleic acid expression construct, comprising: mixing the nucleic acid expression construct with a charged transfection-promoting polypeptide to obtain a stabilized nucleic acid expression construct; in (a) the in vitro degradation of the stabilized nucleic acid expression construct is slowed compared to a nucleic acid expression construct that is not associated with a charged transfection-promoting polypeptide; and (b) The molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct comprises from 1 to 5000 moles of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 72. The method of claim 71, wherein the charged transfection-facilitating polypeptide comprises a poly-L-glutamic acid polypeptide. 73. The method of claim 71, wherein the nucleic acid expression vector has an average molecular length of about 2000 to about 5000 nucleotide base pairs. 74. The method of claim 71, wherein the charged transfection-facilitating polypeptide has an average molecular weight of about 400 to about 30,000 Da. 75. The method of claim 71, wherein the nucleic acid expression vector has an average molecular length of about 5000 nucleotide base pairs and the charged transfection-facilitating polypeptide has an average molecular weight of about 10900 Da. 76. The method of claim 71, wherein the nucleic acid expression construct comprises SeqED#11, SeqID#12, SeqID#13, SeqID#14, SeqID#17, SeqID#18, SeqID#19, SeqID#20, or SeqID#21. 77. The method of claim 71, wherein the molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct is equal to 1200 moles or less of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 78. The method of claim 71, wherein the molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct is equal to 1 mole of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 79. The method of claim 71, wherein the nucleic acid expression construct encodes a gene for growth hormone releasing hormone ("GHRH") or a biologically functional equivalent thereof. 80. The method of claim 79, wherein the encoded GHRH or its biologically functional equivalent has the general formula (SEQ ID #6): -X ₁ -X ₂ -DAIFTNSYRKVL-X ₃ -QLSARKLLQDI-X ₄ -X ₅ -RQQGERNQEQGA-OH Wherein the general formula has the following characteristics: _X is the D- or L-isomer of tyrosine ("Y") or histidine ("H"); _X is the D- or L-isomer of alanine ("A"), valine ("V") or isoleucine ("I"); _X is the D- or L-isomer of alanine ("A") or glycine ("G"); _X is the D- or L-isomer of methionine ("M") or leucine ("L"); _X5 is the D- or L-isomer of serine ("S") or asparagine ("N"); or a combination thereof. 81. The method of claim 71, wherein the nucleic acid expression construct encodes a polypeptide sequence comprising SeqID #1, SeqID #2, SeqID #3, SeqID #4 or SeqID #5. 82. A method of increasing the stability of a nucleic acid expression construct, comprising: mixing the nucleic acid expression construct with a charged transfection-promoting polypeptide to provide a stabilized nucleic acid expression construct; in The in vitro degradation of the stabilized nucleic acid expression construct is slowed down compared to a nucleic acid expression construct not associated with a transfection-promoting polypeptide; Charged transfection-promoting polypeptides include poly-L-glutamic acid polypeptides; The nucleic acid expression construct encodes growth hormone releasing hormone ("GHRH") or a biologically functional equivalent thereof; and The molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct comprises from 1 to 5000 moles of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 83. The method of claim 82, wherein the nucleic acid expression vector has an average molecular length of about 2000 to about 5000 nucleotide base pairs. 84. The method of claim 82, wherein the charged transfection-facilitating polypeptide has an average molecular weight of about 400 to about 30,000 Da. 85. The method of claim 82, wherein the nucleic acid expression vector has an average molecular length of about 5000 nucleotide base pairs and the charged transfection-facilitating polypeptide has an average molecular weight of about 10900 Da. 86. The method of claim 82, wherein the nucleic acid expression construct comprises SeqED#11, SeqID#12, SeqID#13, SeqID#14, SeqID#17, SeqID#18, SeqID#19, SeqID#20, or SeqID#21. 87. The method of claim 82, wherein the molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct is equal to 1200 moles or less of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 88. The method of claim 82, wherein the molar ratio of charged transfection-facilitating polypeptide to nucleic acid expression construct is equal to 1 mole of charged transfection-facilitating polypeptide per mole of nucleic acid expression construct. 89. The method of claim 82, wherein the encoded GHRH or its biologically functional equivalent has the general formula (SEQ ID #6): -X ₁ -X ₂ -DAIFTNSYRKVL-X ₃ -QLSARKLLQDI-X ₄ -X ₅ -RQQGERNQEQGA-OH Wherein the general formula has the following characteristics: _X is the D- or L-isomer of tyrosine ("Y") or histidine ("H"); _X is the D- or L-isomer of alanine ("A"), valine ("V") or isoleucine ("I"); _X is the D- or L-isomer of alanine ("A") or glycine ("G"); _X is the D- or L-isomer of methionine ("M") or leucine ("L"); _X5 is the D- or L-isomer of serine ("S") or asparagine ("N"); or a combination thereof. 90. The method of claim 82, wherein the nucleic acid expression construct encodes a polypeptide sequence comprising SeqID #1, SeqID #2, SeqID #3, SeqID #4, or SeqID #5.

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