Abstract: The present invention pertains to compositions and methods for plasmid-mediated supplementation. The compositions and methods are useful for treatment or prevention of kidney failure, treatment of anemia, and other conditions commonly associated with kidney failure in order to increase survival and improve welfare in subjects with chronic renal failure. Overall, the embodiments of the invention can be accomplished by delivering an isolated nucleic acid expression construct that encodes a GHRH or functional biological equivalent thereof into a tissue of a subject and allowing expression of the encoded gene in the animal. For example, when such a nucleic acid sequence is delivered into the specific cells of the subject, tissue specific constitutive expression is achieved. The embodiments of the invention also encompass delivery of a recombinant GHRH polypeptide or functional biological equivalent thereof.

Abstract: One aspect of the current invention is a method of decreasing an involuntary cull rate in farm animals, wherein the involuntary cull results from infection, disease, morbidity, or mortality. Additionally, milk production, animal welfare, and body condition scores are improved by utilizing methodology that administers the isolated nucleic acid expression construct encoding a GHRH or functional biological equivalent to an animal through a parenteral route of administration. Following a single dose of nucleic acid expression vector, animals are healthier and effects are demonstrated long term without additional administration(s) of the expression construct.

Abstract: One aspect of the current invention is a composition for a modified growth hormone releasing hormone (“GHRH”) or functional biological equivalent thereof. Another aspect of the current invention includes a nucleic acid molecule that encodes the modified GHRH or functional biological equivalent. The modified GHRH can be defined as a biologically active polypeptide that was engineered to contain a distinct amino acid sequence while simultaneously having similar or improved biologically activity when compared to a wild-type GHRH (“wt-GHRH”) polypeptide. Another aspect of the current invention includes a method for delivering the composition of this invention to a subject, wherein the modified GHRH increases the level of growth hormone (“GH”) secretion in a subject. The preferred subject is a human or domesticated animal. Additionally, the modified GHRH composition is resistant to degradation when compared to the wt-GHRH.

Abstract: Transgenes driven by naturally occurring cardiac promoters have relatively low levels of cardiac transgenic gene expression, and have consequently limited the use of cardiac muscle as a target for plasmid mediated gene supplementation. However, by randomly assembling motifs of E-box, MEF-2, TEF-1 and SRE elements, cardiac-specific synthetic promoter recombinant libraries have been produced. By screening hundreds of resultant clones for transcriptional activity both in vitro and in vivo, a few cardiac-specific synthetic promoters were discovered comprising a transcriptional potency that greatly exceeds the transcriptional levels obtained from natural myogenic and viral gene promoters. These promoters are used to direct the expression of desirable genes in nucleic acid expression constructs specifically to cardiac cells.

Abstract: The present invention relates to a modular electrode system, and its use, for facilitating the introduction of a macromolecule into cells of a selected tissue in a body or plant. The modular electrode system comprises a plurality of needle electrodes; a hypodermic needle; an electrical connector that provides a conductive link from a programmable constant-current pulse controller to the plurality of needle electrodes; and a power source. In a preferred embodiment of the present invention, an operator can grasp the plurality of needle electrodes that are mounted on a support structure and firmly insert the them into the selected tissue in a body or plant. The macromolecules are then delivered via the hypodermic needle into the selected tissue. The programmable constant-current pulse controller is activated and constant-current electrical pulse is applied to the plurality of needle electrodes.

Abstract: The present invention pertains to a method for decreasing the body fat proportion, increasing lean body mass (“LBM”), increasing bone density, or improving the rate of bone healing, or all, of a subject. Overall, the embodiments of the invention can be accomplished by delivering a heterologous nucleic acid sequence encoding GHRH or functional biological equivalent thereof into the cells of the subject and allowing expression of the encoded gene to occur while the modified cells are within the subject. For instance, when such a nucleic acid sequence is delivered into the specific cells of the subject tissue specific constitutive expression is achieved. Furthermore, external regulation of the GHRH or functional biological equivalent thereof gene can be accomplished by utilizing inducible promoters that are regulated by molecular switch molecules, which are given to the subject.

Abstract: One aspect of the current invention is an optimized synthetic mammalian expression plasmid (e.g. pAV0201). This new plasmid comprise a therapeutic element, and a replication element. The therapeutic element of the new plasmid comprises a eukaryotic promoter; a 5′ untranslated region (“UTR”); a codon-optimized-eukaryotic therapeutic gene sequence; and a poly adenylation signal. The therapeutic elements of this plasmid are operatively linked and located in a first operatively-linked arrangement. Additionally, the optimized synthetic mammalian expression plasmid comprises replication elements, wherein the replication elements are operatively linked and located in a second operatively-linked arrangement. The replication elements comprise a selectable marker gene promoter, a ribosomal binding site, and an origin of replication.

Abstract: The present invention pertains to compositions and methods for plasmid-mediated supplementation. The compositions and method are useful for retarding the growth of the tumor, and retarding cachexia, wasting, anemia and other effects that are commonly associated in cancer bearing animals. Overall, the embodiments of the invention can be accomplished by delivering an effective amount of a nucleic acid expression construct that encodes a GHRH or functional biological equivalent thereof into a tissue of an animal and allowing expression of the encoded gene in the animal. For example, when such a nucleic acid sequence is delivered into the specific cells of the animal tissue specific constitutive expression is achieved. Furthermore, external regulation of the GHRH or functional biological equivalent thereof gene can be accomplished by utilizing inducible promoters that are regulated by molecular switch molecules, which are given to the animal.

Abstract: The intramuscular electroporated injection of a protease-resistant growth hormone-releasing hormone (“GHRH”) cDNA into rat dams at 16 days of gestation resulted in the enhanced long-term growth of the F1 offspring. The offspring were significantly heavier by one week of age and the difference was sustained to 10 weeks of age. Consistent with their augmented growth, plasma IGF-I concentration of the F1 progeny was increased significantly. The pituitary gland of the offspring was significantly heavier, and contained an increased number of somatotropes (cells producing GH) and lactotrophs (prolactin-secreting cells), and is indicative of an alteration in cell lineages. These unique findings demonstrate that enhanced GHRH expression in pregnant dams can result in intergenerational growth promotion, by altering development of the pituitary gland in the offspring.

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

Abstract: Linear double-stranded DNA fragments containing a promoter, a nucleotide sequence, such as a transgene, preferably non-viral, and a 3′ untranslated region, are delivered to tissue of an animal by direct injection accompanied by electroporation. Long-term expression of the transgene results in prolonged availability of proteins, hormones, or enzymes that may be deficient in the mammal. In addition, the linear fragments increase the safety of the vectors for mammalian gene therapy by avoiding deleterious side effects.

Abstract: Growth is improved by utilizing growth enhancement potential methodology to administer a nucleic acid sequence, such as GHRH or an analog, to a female animal, preferably through a parenteral route of administration. Piglets born from sows injected with DNA encoding GHRH are larger, and effects are demonstrated in subsequent pregnancies without additional administration(s) of the vector.

Abstract: Vectors which establish controlled expression of recombinant GHRH genes within tissues at certain levels. The vector includes a 5′ flanking region which includes necessary sequences for expression of a nucleic acid cassette, a 3′ flanking region including a 3′ UTR and/or 3′ NCR, and a linker which connects the 5′ flanking region to a nucleic acid sequence. The linker has a position for inserting a nucleic acid cassette. The linker does not contain the coding sequence of a gene that the linker is naturally associated with. The 3′ flanking region is 3′ to the position for inserting the nucleic acid cassette.

Abstract: Vectors which establish controlled expression of recombinant GHRH genes within tissues at certain levels. The vector includes a 5′ flanking region which includes necessary sequences for expression of a nucleic acid cassette, a 3′ flanking region including a 3′UTR and/or 3′NCR, and a linker which connects the 5′ flanking region to a nucleic acid sequence. The linker has a position for inserting a nucleic acid cassette. The linker does not contain the coding sequence of a gene that the linker is naturally associated with. The 3′ flanking region is 3′ to the position for inserting the nucleic acid cassette.