Gene delivery vectors with cell type specificity for mesenchymal stem cells

Abstract

Methods and associated materials for transducing mesenchymal stem cells with a desired nucleic acid. Mesenchymal stem cells are a recently discovered kind of stem cell for which suitable transfer vehicles are still desired. Typical gene delivery vehicles such as the adenoviruses or adeno associated viruses have no particular tropism for mesenchymal stem cells. Also disclosed is gene therapy using adenoviruses provided with tropism for mesenchymal stem cells.

1 . A method of delivering at least one nucleic acid sequence of interest to a mesenchymal stem cell, said method comprising: 
 culturing a mesenchymal stem cell in vitro; and    infecting said mesenchymal stem cell with a recombinant adenovirus of subgroup C, said recombinant adenovirus comprising the at least one nucleic acid sequence of interest, and further comprising a capsid, wherein said capsid comprises a fiber protein knob of an adenovirus of subgroup B, so as to introduce said at least one nucleic acid sequence of interest into said cultured mesenchymal stem cell.    
     
     
         2 . The method according to  claim 1 , wherein the recombinant adenovirus of subgroup C comprises a recombinant adenovirus of serotype 5.  
     
     
         3 . The method according to  claim 2 , wherein the fiber protein of an adenovirus of subgroup B comprises a tail of adenovirus serotype 5.  
     
     
         4 . The method according to  claim 1 , wherein the fiber protein knob of an adenovirus of subgroup B provides the recombinant adenovirus with a tropism for mesenchymal stem cells.  
     
     
         5 . The method according to  claim 1 , wherein the fiber knob of an adenovirus of subgroup B is of a serotype selected from the group consisting of serotype 16, serotype 32, serotype 35, serotype 40-S, and serotype 51.  
     
     
         6 . The method according to  claim 1 , wherein the at least one nucleic acid sequence of interest encodes a protein selected from the group consisting of: Factor VIII, Factor IX, β-glucocerebrosidase and mutations thereof, erythropoietin, α-L-iduronidase, iduronate sulphatase, N-sulphatase, N-acetyl α-D-glucosamimidase, α-glucosamine-N-acetyltransferase, N-acetyl-α-D-glucosaminide-6-sulphatase, galactosamine-6 sulphate sulphatase, β-galactosidase, N-acetyl-alactosamine-4-sulphatase, acid ceraminidase, acid sphingomyelinase, galactocerebroside β-galactosidase, arylsuphatase A, adenosine deaminase, α-L-fucosidase, interleukins, angiogenesis stimulating or inhibiting factors such as nitric oxide synthase, vascular endothelial growth factor, C-naturetic peptide, bone morphogenesis protein-2, LIM mineralization protein-1, and angiostatin.  
     
     
         7 . The method according to  claim 1 , wherein the recombinant adenovirus further comprises at least a part of an adenoviral genome having a deletion in a E1 region, said deletion rendering the recombinant adenovirus replication defective.  
     
     
         8 . The method according to  claim 7 , wherein the at least one nucleic acid sequence of interest is incorporated into the E1 region of the recombinant adenovirus genome.  
     
     
         9 . The method according to  claim 1 , wherein the at least one nucleic acid of interest is operably linked to a promoter.  
     
     
         10 . The method according to  claim 1 , further comprising stably integrating the at least one nucleic acid of interest into the mesenchymal stem cell genome.  
     
     
         11 . The method according to  claim 1 , further comprising replicating the recombinant adenovirus in a PER.C6 cell culture.  
     
     
         12 . The method according to  claim 1 , wherein the at least one nucleic acid sequence of interest is incorporated into the E3 region of the recombinant adenovirus genome.  
     
     
         13 . A mesenchymal stem cell obtained by a process comprising: 
 culturing a mesenchymal stem cell in vitro; and    infecting said mesenchymal stem cell with a recombinant adenovirus of subgroup C, said recombinant adenovirus comprising at least a part of an adenoviral genome, said at least part of an adenoviral genome comprising a nucleic acid sequence of interest, and said recombinant adenovirus further comprising a capsid, wherein said capsid comprises a fiber protein knob of an adenovirus of subgroup B.    
     
     
         14 . The mesenchymal stem cell of  claim 13 , wherein the recombinant adenovirus of subgroup C comprises a recombinant adenovirus of serotype 5.  
     
     
         15 . The mesenchymal stem cell of  claim 14 , wherein the fiber protein of an adenovirus of subgroup B comprises a tail of adenovirus serotype 5 amino acid sequence.  
     
     
         16 . The mesenchymal stem cell of  claim 13 , wherein the knob of a fiber protein of an adenovirus of subgroup B is of a serotype selected from the group consisting of serotype 16, serotype 32, serotype 35, serotype 40-S, and serotype 51.  
     
     
         17 . The mesenchymal stem cell of  claim 13 , wherein the at least one exogenous nucleic acid sequence of interest encodes for a protein selected from the group consisting of: Factor VIII, Factor IX, β-glucocerebrosidase and mutations thereof, erythropoietin, α-L-iduronidase, iduronate sulphatase, N-sulphatase, N-acetyl α-D-glucosamimidase, α-glucosamine-N-acetyltransferase, N-acetyl-α-D-glucosaminide-6-sulphatase, galactosamine-6 sulphate sulphatase, β-galactosidase, N-acetyl-alactosamine-4-sulphatase, acid ceraminidase, acid sphingomyelinase, galactocerebroside β-galactosidase, arylsuphatase A, adenosine deaminase, α-L-fucosidase, interleukins, angiogenesis stimulating or inhibiting factors such as nitric oxide synthase, vascular endothelial growth factor, C-naturetic peptide, bone morphogenesis protein-2, LIM mineralization protein-1, and angiostatin.  
     
     
         18 . A mesenchymal stem cell infected with a recombinant adenovirus of subgroup C, wherein said recombinant adenovirus of subgroup C comprises a capsid, said capsid comprising a fiber protein knob of an adenovirus of subgroup B, and wherein said recombinant adenovirus further comprises a nucleic acid sequence of interest.  
     
     
         19 . The mesenchymal stem cell of  claim 18 , wherein said recombinant adenovirus is internalized inside an intracellular vesicle of said mesenchymal stem cell.