Canine specific growth hormone releasing hormone

Abstract

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

1 . A composition comprising a nucleic acid expression construct encoding a canine specific growth hormone releasing hormone (“dGHRH”) or fragments thereof, having a sequence at least 95% identical to SEQ ID NO:4: 
       
         
           
                 
                 
                 
               
                     
                     X   1   X   2 DAIFTNSYRKVL X   3 QLSARKLLQDI X   4   X   5 RQQGERN R EQGA 
                     
                 
             
                
               
            
           
         
       
       wherein:
 X1 is a D- or L-isomer of an amino acid selected from the group consisting of tyrosine (“Y”), or histidine (“H”); X 2  is a D- or L-isomer of an amino acid selected from the group consisting of alanine (“A”), valine (“V”), or isoleucine (“I”); X 3  is a D- or L-isomer of an amino acid selected from the group consisting of alanine (“A”) or glycine (“G”); X 4  is a D- or L-isomer of an amino acid selected from the group consisting of methionine (“M”), or leucine (“L”); X 5  is a D- or L-isomer of an amino acid selected from the group consisting of serine (“S”) or asparagines (“N”). 
 
     
     
         2 . The composition of  claim 1 , wherein the dGHRH increases growth hormone (“GH”) in a recipient subject. 
     
     
         3 . The composition of  claim 1 , wherein the dGHRH improves hematological parameters in a recipient subject. 
     
     
         4 . The composition of  claim 1 , wherein the dGHRH increases red blood cell values in a recipient subject. 
     
     
         5 . The composition of  claim 1 , wherein the dGHRH increases hemoglobin values in a recipient subject. 
     
     
         6 . The composition of  claim 1 , wherein the dGHRH increases mean corpuscular hemoglobin values in a recipient subject. 
     
     
         7 . The composition of  claim 1 , wherein the nucleic acid expression construct further comprises:
 (a) a synthetic or eukaryotic promoter;   (b) a poly-adenylation signal;   (c) a selectable marker gene promoter;   (d) a ribosomal binding site;   (e) a selectable marker gene sequence; and   (f) an origin of replication;   
       wherein, the synthetic or eukaryotic promoter, the nucleic acid sequence encoding the dGHRH or functional biological equivalent thereof, and the poly adenylation signal comprise therapeutic elements of the nucleic acid expression construct; the therapeutic elements are operatively linked and located in a first operatively-linked arrangement; the selectable marker gene promoter, the ribosomal binding site, the selectable marker gene sequence, and the origin of replication comprise replication elements of the nucleic acid expression construct; the replication elements are operatively linked and located in a second operatively-linked arrangement; the first-operatively-linked arrangement and the second-operatively-linked arrangement comprise a circular structure of the nucleic acid expression construct; and the nucleic acid expression construct is utilized for plasmid mediated gene supplementation. 
     
     
         8 . The composition of  claim 7 , further comprising a 5′ untranslated region (“UTR”) operatively linked to the first operatively-linked arrangement. 
     
     
         9 . The composition of  claim 7 , wherein the 3′ untranslated region (“UTR”) comprises a portion of a human growth hormone 3′UTR. 
     
     
         10 . A nucleic acid expression construct pAV0221 comprising a sequence at least 95% identical to SEQ ID D NO: 5. 
     
     
         11 . A nucleic acid expression construct pAV00215 comprising a sequence at least 95% identical to SEQ ID NO: 6. 
     
     
         12 . A method of increasing growth hormone (“GH”) values in a subject comprising delivering to the subject an isolated composition of canine specific growth hormone releasing hormone (“dGHRH”) or fragments thereof, having a sequence at least 95% identical to SEQ ID NO: 4: 
       
         
           
                 
                 
                 
               
                     
                     X   1   X   2 DAIFTNSYRKVL X   3 QLSARKLLQDI X   4   X   5 RQQGERN R EQGA 
                     
                 
             
                
               
            
           
         
       
       wherein:
 X1 is a D- or L-isomer of an amino acid selected from the group consisting of tyrosine (“Y”), or histidine (“H”); X2 is a D- or L-isomer of an amino acid selected from the group consisting of alanine (“A”), valine (“V”), or isoleucine (“I”); X3 is a D- or L-isomer of an amino acid selected from the group consisting of alanine (“A”) or glycine (“G”); X4 is a D- or L-isomer of an amino acid selected from the group consisting of methionine (“M”), or leucine (“L”); X5 is a D- or L-isomer of an amino acid selected from the group consisting of serine (“S”) or asparagines (“N”). 
 
     
     
         13 . The method of  claim 12 , wherein the recombinant dGHRH is a biologically active polypeptide, and the recombinant functional biological equivalent of dGHRH is a polypeptide that has been engineered to contain a distinct amino acid sequence while simultaneously having similar or improved biologically activity when compared to the dGHRH polypeptide. 
     
     
         14 . The method of  claim 12 , wherein increasing hematological parameters in the subject having the delivered recombinant dGHRH nucleic acid expression construct reflects increasing GH values. 
     
     
         15 . The method of  claim 14 , wherein the hematological parameter is red blood cell count, hemoglobin concentration, or mean corpuscular hemoglobin. 
     
     
         16 . A method of increasing growth hormone (“GH”) values in a subject comprising: delivering into cells of the subject a nucleic acid expression construct that encodes a canine specific growth-hormone-releasing-hormone (“dGHRH”) or fragments thereof, having a sequence at least 95% identical to SEQ ID NO: 5: 
       
         
           
                 
                 
                 
               
                     
                     X   1   X   2 DAIFTNSYRKVL X   3 QLSARKLLQDI X   4   X   5 RQQGERN R EQGA 
                     
                 
             
                
               
            
           
         
       
       wherein:
 X1 is a D- or L-isomer of an amino acid selected from the group consisting of tyrosine (“Y”), or histidine (“H”); X2 is a D- or L-isomer of an amino acid selected from the group consisting of alanine (“A”), valine (“V”), or isoleucine (“I”); X3 is a D- or L-isomer of an amino acid selected from the group consisting of alanine (“A”) or glycine (“G”); X4 is a D- or L-isomer of an amino acid selected from the group consisting of methionine (“M”), or leucine (“L”); X5 is a D- or L-isomer of an amino acid selected from the group consisting of serine (“S”) or asparagines (“N”). 
 
     
     
         17 . The method of  claim 16 , wherein delivering into the cells of the subject the nucleic acid expression construct is via electroporation. 
     
     
         18 . The method of  claim 16 , wherein the cells of the subject are somatic cells, stem cells, or germ cells. 
     
     
         19 . The method of  claim 16 , wherein the nucleic acid expression construct is SEQ ID NO: 5 or SEQ ID NO: 6. 
     
     
         20 . The method of  claim 16 , wherein the nucleic acid expression construct further comprises a transfection-facilitating polypeptide. 
     
     
         21 . The method of  claim 20 , wherein the transfection-facilitating polypeptide comprises a charged polypeptide. 
     
     
         22 . The method of  claim 20 , wherein the transfection-facilitating polypeptide comprises poly-L-glutamate. 
     
     
         23 . The method of  claim 16 , wherein the dGHRH is expressed in tissue-specific cells of the subject. 
     
     
         24 . The method of  claim 23 , wherein the tissue specific cells of the subject comprises muscle cells. 
     
     
         25 . The method of  claim 16 , wherein increasing hematological parameters in the subject having the delivered nucleic acid expression construct reflects increasing GH values. 
     
     
         26 . The method of  claim 25 , wherein the hematological parameter is red blood cell count, hemoglobin concentration, or mean corpuscular hemoglobin. 
     
     
         27 . A composition comprising an isolated canine specific growth hormone releasing hormone (“dGHRH”) or fragments thereof, having a sequence at least 95% identical to SEQ ID NO: 4: 
       
         
           
                 
                 
                 
               
                     
                     X   1   X   2 DAIFTNSYRKVL X   3 QLSARKLLQDI X   4   X   5 RQQGERN R EQGA 
                     
                 
             
                
               
            
           
         
       
       wherein:
 X1 is a D- or L-isomer of an amino acid selected from the group consisting of tyrosine (“Y”), or histidine (“H”); X 2  is a D- or L-isomer of an amino acid selected from the group consisting of alanine (“A”), valine (“V”), or isoleucine (“I”); X 3  is a D- or L-isomer of an amino acid selected from the group consisting of alanine (“A”) or glycine (“G”); X 4  is a D- or L-isomer of an amino acid selected from the group consisting of methionine (“M”), or leucine (“L”); X 5  is a D- or L-isomer of an amino acid selected from the group consisting of serine (“S”) or asparagines (“N”); and the isolated dGHRH is a biologically active polypeptide that has been engineered to contain a distinct amino acid sequence. 
 
     
     
         28 . The composition of  claim 27 , wherein the isolated dGHRH increases growth hormone (“GH”) in a recipient subject. 
     
     
         29 . The composition of  claim 27 , wherein the isolated dGHRH improves hematological parameters in a recipient subject. 
     
     
         30 . The composition of  claim 27 , wherein the isolated dGHRH thereof increases red blood cell values in a recipient subject. 
     
     
         31 . The composition of  claim 27 , wherein the isolated dGHRH thereof increases hemoglobin values in a recipient subject. 
     
     
         32 . The composition of  claim 27 , wherein the isolated dGHRH increases mean corpuscular hemoglobin values in a recipient subject.