Growth hormone releasing hormone expression system and methods of use, including use in animals

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.

We claim:  
     
         1 . A vector for expression of a nucleic acid sequence in a cell, comprising: 
 a nucleic acid cassette containing a nucleotide sequence encoding GHRH;    a 5′ flanking region including one or more sequences necessary for expression of said nucleic acid cassette, wherein said sequences include a promoter;    a linker connecting said 5′ flanking region to a nucleic acid, said linker having a position for inserting said nucleic acid cassette, wherein said linker lacks the coding sequence of a gene with which it is naturally associated; and    a 3′ flanking region, including a 3′ UTR or a 3′ NCR or both, wherein said 3′ flanking region is 3′ to said position for inserting said nucleic acid cassette, and wherein said 3′ flanking region comprises a sequence from a 3′-UTR.    
     
     
         2 . The vector of  claim 1 , wherein said GHRH is human GHRH.  
     
     
         3 . The vector of  claim 2 , wherein said nucleotide sequence encoding for human GHRH is a synthetic sequence.  
     
     
         4 . The vector of  claim 3 , wherein said nucleotide sequence encoding for human GHRH has the sequence of SEQ ID NO. 2.  
     
     
         5 . The vector of  claim 1 , wherein said promoter is a promoter from a skeletal α-actin gene.  
     
     
         6 . The vector of  claim 5 , wherein said promoter from a skeletal α-actin gene is from a chicken.  
     
     
         7 . The vector of  claim 5 , wherein said promoter from a skeletal α-actin gene is from a human.  
     
     
         8 . The vector of  claim 1 , wherein said 3′-UTR is a growth hormone 3′-UTR.  
     
     
         9 . The vector of  claim 8 , wherein said growth hormone 3′-UTR is from a human growth hormone gene.  
     
     
         10 . The vector of  claim 8 , wherein an ALU repeat or ALU repeat-like sequence is deleted from said 3′ UTR.  
     
     
         11 . The vector of  claim 1 , wherein said GHRH is human GHRH, said promoter is from a chicken skeletal α-actin gene, and said 3′-UTR is from a human growth hormone gene.  
     
     
         12 . The vector of  claim 1 , wherein said 5′ flanking region or said 3′ flanking region or both regulates expression of said nucleic acid cassette predominately in a specific tissue.  
     
     
         13 . The vector of  claim 12 , wherein said specific tissue is myogenic.  
     
     
         14 . The vector of  claim 1 , wherein said 5′ flanking region includes a promoter, a TATA box, a Cap site and a first intron and intron/exon boundary in appropriate relationship for expression of said nucleic acid cassette.  
     
     
         15 . The vector of  claim 14 , wherein said 5′ flanking region further comprises a 5′ mRNA leader sequence inserted between said promoter and said nucleic acid cassette.  
     
     
         16 . The vector of  claim 1 , wherein said vector further comprises an intron/5′ UTR from a chicken skeletal α-actin gene.  
     
     
         17 . The vector of  claim 1 , wherein said vector further comprises an antibiotic resistance gene.  
     
     
         18 . The vector of  claim 1 , wherein said vector comprises a nucleotide sequence which is the same as the nucleotide sequence of plasmid PSK-GHRH.  
     
     
         19 . A formulation for delivery and expression of a human GHRH gene in a cell, said formulation comprising a vector of  claim 1  in a solution having between  0 . 5 % and 50% PVP.  
     
     
         20 . The formulation of  claim 19 , wherein said solution includes about 5% PVP.  
     
     
         21 . A transgenic animal having a plurality of cells containing the vector of  claim 1 .  
     
     
         22 . The transgenic animal of  claim 21 , wherein said cell is a germ or somatic cell.  
     
     
         23 . A cell transformed with a vector of  claim 1 .  
     
     
         24 . The transformed cell of  claim 23 , wherein said cell is myogenic.  
     
     
         25 . A method for transfection of a cell in situ, comprising the step of contacting said cell with a vector of  claim 1  for sufficient time to transfect said cell.  
     
     
         26 . The method of  claim 25 , wherein transfection of said cell is performed in vivo.  
     
     
         27 . The method of  claim 26 , wherein said contacting is performed in the presence of an about 5% PVP solution.  
     
     
         28 . The method of  claim 25 , wherein transfection of said cell is performed ex vivo, further comprising the steps of cotransfecting said vector with a selectable marker and selecting the transformed cells.  
     
     
         29 . A method for delivery and expression of a GHRH gene in a plurality of cells, comprising the steps of: 
 (a) transfecting said plurality of cells with a vector of  claim 1;  and    (b) incubating said plurality of cells under conditions allowing expression of a nucleic acid sequence in said vector, wherein said nucleic acid sequence encodes GHRH.    
     
     
         30 . The method of  claim 29 , wherein said GHRH is hGHRH and said cells are human cells.  
     
     
         31 . The method of  claim 30 , wherein said contacting is performed in the presence of an about 5% PVP solution.  
     
     
         32 . A method for treating a disease or condition, comprising the steps of transfecting a cell in situ with a vector of  claim 1 .  
     
     
         33 . The method of  claim 32 , wherein said disease or condition is a localized disease or condition.  
     
     
         34 . The method of  claim 32 , wherein said disease of condition is a systemic disease or condition.  
     
     
         35 . The method of  claim 32 , wherein said disease or condition to be treated is selected from the group consisting of osteoporosis, cachexia, and growth disorders.  
     
     
         36 . A method of expressing growth hormone releasing (GHRH) in a non-human vertebrate animal comprising the step of: inserting a DNA carrier vehicle containing a gene sequence encoding a growth hormone releasing hormone polypeptide sequence operatively linked to a vertebrate gene promoter into said non-human vertebrate animal tissue under conditions where said gene is expressed and produces growth hormone releasing hormone.  
     
     
         37 . The method of  claim 36  wherein said gene sequence encodes for a growth hormone releasing hormone having one of the following sequences: SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, or SEQ ID NO.11.  
     
     
         38 . The method of  claim 36  wherein said vertebrate animal is one of the following species; porcine, bovine, equine, canine, feline, caprine, avian (chicken, turkey, duck), ovine or fish.  
     
     
         39 . The method of  claim 36  wherein the gene sequence in said DNA carrier vehicle contains no intervening sequences.  
     
     
         40 . The method of  claim 36 , wherein said promoter is from a skeletal a-actin gene.  
     
     
         41 . The method of  claim 36 , wherein said DNA carrier vehicle is injected into said animal muscle.  
     
     
         42 . The method of  claim 36 , wherein said DNA carrier vehicle is a plasmid DNA vector capable of infecting said vertebrate animals in various tissues.  
     
     
         43 . The method of  claim 36 , wherein said DNA carrier vehicle is an adenovirus or adeno-associated virus capable of infecting said vertebrate animals in various tissues.  
     
     
         44 . The method of  claim 43 , wherein the promoter—GHRHcDNA-3′ UTR is incorporated into said adeno-associated virus.  
     
     
         45 . The method of  claim 36 , wherein said vectors encode for an Arg-Arg sequence before a tyrosine or a histidine.  
     
     
         46 . The method of  claim 36 , wherein said DNA carrier vehicle includes a gene switch.  
     
     
         47 . The method of  claim 36 , wherein said gene sequence is a chimeric synthetic cDNA encoding GHRH comprising a mouse specific fragment and a species specific fragment and wherein the mouse specific fragment contains the first 45 nucleotides and encodes the first 15 amino acids of the mouse GHRH, and said mouse specific fragment is fused in frame with the species-specific fragment contains 87 nucleotides and encodes the 16th to 44th amino acids of a species-specific GHRH, said chimeric sequence providing resistance against dipeptidases.  
     
     
         48 . The method of  claim 47 , wherein said species-specific fragment for GHRH encodes a polypeptide from one of the following animal species; porcine, bovine, equine, canine, feline, caprine, avian (chicken, turkey, duck) ovine or fish.  
     
     
         49 . The method of  claim 47 , wherein said species-specific fragment of GHRH encodes DNA sequence encodes for one of the following GHRH polypeptides: SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, or SEQ ID NO.11.