Modified pituitary gland development in offspring from expectant mother animals treated with growth hormone releasing hormone therapy

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 FI 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 FI 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.

1 . A method of changing the pituitary lineage in an offspring from a female subject comprising:
 delivering a nucleic acid expression construct into cells of the female subject; wherein,
 the delivering is completed prior to or during a gestation period of the offspring; 
 the nucleic acid expression construct comprises a promoter; a nucleotide sequence a growth-hormone-releasing-hormone (“GHRH”) or functional biological equivalent thereof; and a 3′ untranslated region; and 
 delivering is completed under conditions wherein expression of the nucleotide sequence results in the changing the pituitary lineage in the offspring; 
   
     
     
         2 . The method of  claim 1 , wherein the delivering of the nucleic acid expression construct into the cells of the female subject comprises electroporation. 
     
     
         3 . The method of  claim 1 , wherein the delivering of the nucleic acid expression construct into the cells of the female subject comprises use of a viral vector, a carrier, a parenteral route, or a combination thereof. 
     
     
         4 . The method of  claim 1 , wherein the cells of the female subject comprise somatic cells, stem cells, or germ cells. 
     
     
         5 . The method of  claim 1 , wherein the promoter comprises a synthetic myogenic promoter. 
     
     
         6 . The method of  claim 1 , wherein the 3′ untranslated region comprises a human growth hormone (“hGH”) 3′ untranslated region (SEQ ID#8). 
     
     
         7 . The method of  claim 1 , wherein the nucleic acid expression construct comprises pSP-HV-GHRH (SEQ ID#11), or its functional nucleic acid equivalent. 
     
     
         8 . The method of  claim 1 , wherein the nucleic acid expression construct is a TI-GHRH plasmid (SEQ ID#12), TV-GHRH Plasmid (SEQ ID#13), 15/27/28 GHRH plasmid (SEQ ID#14), pSP-wt-GHRH plasmid (SEQ ID#15), or its functional nucleic acid equivalent. 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein the delivering into the cells of the female subject the nucleic acid expression construct initiates expression of the encoded GHRH or a functional biological equivalent thereof. 
     
     
         11 . The method of  claim 1 , wherein the encoded GHRH or functional biological equivalent thereof comprise expression in tissue specific cells of the female subject. 
     
     
         12 . The method of  claim 1 , wherein the tissue specific cells of the female subject comprise muscle cells. 
     
     
         13 . The method of  claim 1 , wherein the nucleic acid expression construct further comprises a transfection-facilitating polypeptide. 
     
     
         14 . The method of  claim 13 , wherein the transfection-facilitating polypeptide comprises a charged polypeptide. 
     
     
         15 . The method of  claim 13 , wherein the transfection-facilitating polypeptide comprises poly-L-glutamate. 
     
     
         16 . The method of  claim 1 , wherein the nucleic acid sequence encodes a growth-hormone-releasing-hormone (“GHRH”) or a functional biological equivalent thereof, wherein the GHRH is a biologically active polypeptide; and wherein the functional biological equivalent of GHRH is a polypeptide that has been engineered to contain a distinct amino acid sequence while simultaneously having similar or improved biologically activity when compared to the GHRH polypeptide. 
     
     
         17 . The method of  claim 1 , wherein the nucleic acid sequence encodes a growth-hormone-releasing-hormone (“GHRH”) or a functional biological equivalent thereof, and wherein the GHRH or the functional biological equivalent comprises an amino acid formula (SEQ ID No: 6):
 -X- 1 -X 2 -DAIFTNSYRKVL-X 3 -QLSARKLLQDI-X 4 -X 5 -RQQGERNQEQGA-OH   wherein the formula has the following characteristics:   X 1  is a D- or L-isomer of the amino acid tyrosine (“Y”), or histidine (“H”);   X 2  is a D- or L-isomer of the amino acid alanine (“A”), valine (“V”), or isoleucine (“I”);   X 3  is a D- or L-isomer of the amino acid alanine (“A”) or glycine (“G”);   X 4  is a D- or L-isomer of the amino acid methionine (“M”), or leucine (“L”);   X 5  is a D- or L-isomer of the amino acid serine (“S”) or asparagine (“N”).   
     
     
         18 . The method of  claim 1 , wherein the nucleic acid sequence encodes a growth-hormone-releasing-hormone (“GHRH”) or a functional biological equivalent thereof, that facilitates growth hormone (“GH”) secretion in the female subject. 
     
     
         19 . The method of  claim 1 , wherein the nucleic acid expression construct is introduced into the female subject in a single administration. 
     
     
         20 . The method of  claim 1 , wherein delivering of the nucleic acid expression construct into cells of the female subject occurs during the third trimester of gestation of the offspring. 
     
     
         21 . The method of  claim 1 , further comprising the step of administering to the female subject a ligand for a growth hormone secretagogue receptor. 
     
     
         22 . The method of  claim 21 , wherein the ligand is administered orally. 
     
     
         23 . (canceled) 
     
     
         24 . The method of  claim 5 , wherein the delivering of the nucleic acid expression construct into the cells of the female subject comprises electroporation. 
     
     
         25 . The method of  claim 5 , wherein the delivering of the nucleic acid expression construct into the cells of the female subject comprises use of a viral vector, a carrier, a parenteral route, or a combination thereof. 
     
     
         26 . The method of  claim 5 , wherein the cells of the female subject comprise somatic cells, stem cells, or germ cells. 
     
     
         27 . The method of  claim 5 , wherein the 3′ untranslated region comprises a human growth hormone (“hGH”) 3′ untranslated region (SEQ ID#8). 
     
     
         28 . The method of  claim 5 , wherein the nucleic acid expression construct comprises pSP-HV-GHRH (SEQ ID#11), or its functional nucleic acid equivalent. 
     
     
         29 . The method of  claim 5 , wherein the nucleic acid expression construct is a TI-GHRH plasmid (SEQ ID#12), TV-GHRH Plasmid (SEQ ID#13), 15/27/28 GHRH plasmid (SEQ ID#14), pSP-wt-GHRH plasmid (SEQ ID#15), or its functional nucleic acid equivalent. 
     
     
         30 . The method of  claim 5 , wherein the delivering into the cells of the female subject the nucleic acid expression construct initiates expression of the encoded GHRH or a functional biological equivalent thereof. 
     
     
         31 - 32 . (canceled) 
     
     
         33 . The method of  claim 5 , wherein the nucleic acid expression construct further comprises, an addition of a transfection-facilitating polypeptide with nucleic acid expression construct. 
     
     
         34 . The method of  claim 33 , wherein the transfection-facilitating polypeptide comprises a charged polypeptide. 
     
     
         35 . The method of  claim 33 , wherein the transfection-facilitating polypeptide comprises poly-L-glutamate. 
     
     
         36 . The method of  claim 5 , wherein the nucleic acid sequence encodes a growth-hormone-releasing-hormone (“GHRH”) or a functional biological equivalent thereof, wherein the GHRH is a biologically active polypeptide; and wherein the functional biological equivalent of GHRH is a polypeptide that has been engineered to contain a distinct amino acid sequence while simultaneously having similar or improved biologically activity when compared to the GHRH polypeptide. 
     
     
         37 . The method of  claim 5 , wherein the nucleic acid sequence encodes a growth-hormone-releasing-hormone (“GHRH”) or a functional biological equivalent thereof, and wherein the GHRH or the functional biological equivalent comprises an amino acid formula (SEQ ID No: 6):
 -X- 1 -X 2 -DAIFTNSYRKVL-X 3 -QLSARKLLQDI-X 4 -X 5 -RQQGERNQEQGA-OH   wherein the formula has the following characteristics:   X 1  is a D- or L-isomer of the amino acid tyrosine (“Y”), or histidine (“H”);   X 2  is a D- or L-isomer of the amino acid alanine (“A”), valine (“V”), or isoleucine (“I”);   X 3  is a D- or L-isomer of the amino acid alanine (“A”) or glycine (“G”);   X 4  is a D- or L-isomer of the amino acid methionine (“M”), or leucine (“L”);   X 5  is a D- or L-isomer of the amino acid serine (“S”) or asparagine (“N”).   
     
     
         38 . The method of  claim 5 , wherein the nucleic acid sequence encodes a growth-hormone-releasing-hormone (“GHRH”) or a functional biological equivalent thereof, that facilitates growth hormone (“GH”) secretion in the female subject. 
     
     
         39 . The method of  claim 5 , wherein the nucleic acid expression construct is introduced into the female subject in a single administration. 
     
     
         40 . The method of  claim 5 , wherein delivering of the nucleic acid expression construct into cells of the female subject occurs during the third trimester of gestation of the offspring. 
     
     
         41 . The method of  claim 5 , further comprising the step of administering to the female subject a ligand for a growth hormone secretagogue receptor. 
     
     
         42 . The method of  claim 5 , wherein the ligand is administered orally. 
     
     
         43 - 84 . (canceled)