Viral vectors for the treatment of diabetes

Abstract

They are provided gene constructs comprising a nucleotide sequence encoding the Insulin-like growth factor 1 (IGF-1) of a mammal; and target sequences of a microRNA of a tissue where the expression of IGF-1 is wanted to be prevented, wherein the sequences (a) and (b) are operationally linked to a promoter of ubiquitous expression. Also provided are expression vectors comprising the gene construct and pharmaceutical compositions comprising them. They are useful in the treatment and/or prevention of diabetes mellitus in mammals, wherein a dysfunction and/or a loss of the beta-cells of the islets of Langerhans is present.

1 . A gene construct comprising:
 (a) a nucleotide sequence encoding the Insulin-like growth factor 1 (IGF-1) of a mammal; and   (b) at least one target sequence of a microRNA of a tissue where the expression of IGF-1 is to be prevented, wherein the sequences (a) and (b) are operationally linked to a ubiquitous promoter.   
     
     
         2 . The gene construct according to  claim 1 , wherein the nucleotide sequence encoding the IGF-1 protein is selected from a nucleotide sequence encoding the human IGF-1 protein, which comprises an amino acid sequence selected from the group consisting of sequences having at least 70% identity with SEQ ID NOs: 23 to 27. 
     
     
         3 . The gene construct according to  claim 1 , wherein the nucleotide sequence encoding the IGF-1 of a mammal, is selected from the group consisting of sequences having at least 70% identity with SEQ ID NOs: 3 to 7. 
     
     
         4 . The gene construct according to  claim 1 , wherein the target sequence of a microRNA is selected from a group consisting of sequences SEQ ID NOs: 8 to 20, 22, 93, 94 and 95 and/or combinations thereof. 
     
     
         5 . The gene construct according to  claim 1 , wherein the gene construct comprises at least one target sequence of the microRNA-122a and at least one target sequence of the microRNA-1. 
     
     
         6 . The gene construct according to  claim 1 , wherein the gene construct comprises four copies of the target sequence or of the target sequences of a microRNA. 
     
     
         7 . The gene construct according to  claim 1 , wherein the promoter is a constitutive promoter. 
     
     
         8 . The gene construct according  claim 7 , wherein the promoter is the CAG promoter. 
     
     
         9 . An expression vector that comprises the gene construct comprising:
 (a) a nucleotide sequence encoding the Insulin-like growth factor 1 (IGF-1) of a mammal; and   (b) at least one target sequence of a microRNA of a tissue where the expression of IGF-1 is to be prevented, wherein the sequences (a) and (b) are operationally linked to a ubiquitous promoter.   
     
     
         10 . The vector according to  claim 9 , characterized for being a viral vector selected from the group consisting of adenoviral vectors, adeno associated vectors, retroviral vectors, and lentiviral vectors. 
     
     
         11 . The vector according to  claim 9 , characterized for being an adeno associated viral vector selected from the group consisting of adeno associated viral vector of serotype 6, adeno associated viral vector of serotype 7, adeno associated viral vector of serotype 8, adeno associated viral vector of serotype 9, adeno associated viral vector of serotype 10, adeno associated viral vector of serotype 11, adeno associated viral vector of serotype rh8, and adeno associated viral vector of serotype rh10. 
     
     
         12 . (canceled) 
     
     
         13 . The vector according to  claim 9 , which is for the expression of the gene construct in the pancreas. 
     
     
         14 . (canceled) 
     
     
         15 . A method of treatment and/or prevention of diabetes comprising administering a therapeutically effective amount of the vector of  claim 9 , wherein a dysfunction and/or a loss of the beta-cells of the islets of Langerhans is present. 
     
     
         16 . The method of  claim 15  wherein the diabetes is type 1 diabetes mellitus. 
     
     
         17 . A pharmaceutical composition comprising a therapeutically effective amount of the vector of  claim 9  and one or more pharmaceutically acceptable excipients or vehicles. 
     
     
         18 . An adeno-associated viral (AAV) vector comprising a gene construct comprising:
 (a) a nucleotide sequence encoding the Insulin-like growth factor 1 (IGF-1) of a mammal; and   (b) at least one target sequence of a microRNA of a tissue where the expression of IGF-1 is to be prevented, wherein the sequences (a) and (b) are operationally linked to a ubiquitous promoter.   
     
     
         19 . The AAV vector of  claim 18 , wherein the at least one target sequence of a microRNA is selected from sequences that bind to microRNAs expressed in heart and/or liver. 
     
     
         20 . The AAV vector of  claim 18 , characterized as AAV vector selected from the group consisting of adeno associated viral vector of serotype 6, adeno associated viral vector of serotype 7, adeno associated viral vector of serotype 8, adeno associated viral vector of serotype 9, adeno associated viral vector of serotype 10, adeno associated viral vector of serotype 11, adeno associated viral vector of serotype rh8, and adeno associated viral vector of serotype rh10. 
     
     
         21 . A method of treatment and/or prevention of diabetes comprising administering a therapeutically effective amount of the AAV vector of  claim 18 , preferably diabetes mellitus in mammals, wherein a dysfunction and/or a loss of the beta-cells of the islets of Langerhans is present. 
     
     
         22 . A method of obtaining an AAV vector as defined in  claim 18  comprising:
 (a) providing a cell with:
 (i) a gene construct comprising (a) a nucleotide sequence encoding the Insulin-like growth factor 1 (IGF-1) of a mammal and (b) at least one target sequence of a microRNA of a tissue where the expression of IGF-1 is to be prevented, wherein the sequences (a) and (b) are operationally linked to a ubiquitous promoter, flanked by AAV ITRs; 
 (ii) cap and rep proteins of adeno associated virus; and 
 (iii) adequate viral proteins for the replication of AAV; 
 
 (b) cultivating the cell in suitable conditions to produce the AAV assembly; and 
 (c) purifying the AAV vector produced by the cell.