Genetic modification of primate hemopoietic repopulating stem cells

Abstract

Genetic modification of pluripotent hemopoietic stem cells of primates (P-PHSC) by transduction of P-PHSC with a recombinant adeno-associated virus (AAV). The genome of the recombinant AAV comprises a DNA sequence flanked by the inverted terminal repeats (ITR) of AAV. The DNA sequence will normally comprise regulatory sequences that are functional in hemopoietic cells and, controlled by these regulatory sequences, a sequence coding for a protein or RNA with a therapeutic property when introduced into hemopoietic cells. Preferred examples of DNA sequences are the human lysosomal glucocerebrosidase gene, a globin gene from the human β-globin gene cluster, a DNA sequence encoding an RNA or protein with anti-viral activity, the α1-antitrypsin gene and the human multidrug resistance gene I (MDRI). The invention provides for effective gene therapy with PHSC of primates, particularly humans.

We claim:  
     
         1 . A primate pluripotent hemopoietic stem cell (P-PHSC), said P-PHSC produced by a process of genetically modifying said P-PHSC, said process comprising: 
 harvesting P-PHSC;    after said harvesting, culturing said harvested P-PHSC in a culture medium allowing for proliferation of said P-PHSC; and    after said culturing, introducing a recombinant adeno-associated virus (rAAV)-vector into said cultured P-PHSC to genetically modify said cultured P-PHSC.    
     
     
         2 . The P-PHSC of  claim 1  wherein said rAAV-vector comprises a sequence encoding a protein of interest flanked by AAV inverted terminal repeats (ITRs).  
     
     
         3 . The P-PHSC of  claim 1  wherein said rAAV-vector does not comprise a promoter.  
     
     
         4 . The P-PHSC of  claim 1  wherein said rAAV-vector comprises a promoter not derived from B19 parvovirus.  
     
     
         5 . The P-PHSC of  claim 4  wherein said rAAV-vector comprises a functional part of the β-globin promoter or a functional analog thereof.  
     
     
         6 . The P-PHSC of  claim 4  wherein said rAAV-vector comprises a herpes simplex virus thymidine kinase promoter or a functional analog thereof.  
     
     
         7 . The P-PHSC of  claim 4  wherein said rAAV-vector comprises a ΔMo+PyF101 Long Terminal Repeat promoter or a functional analog thereof.  
     
     
         8 . A cell derived from the P-PHSC of  claim 1 .  
     
     
         9 . A cell derived from the P-PHSC of  claim 2 .  
     
     
         10 . A cell derived from the P-PHSC of  claim 3 .  
     
     
         11 . A cell derived from the P-PHSC of  claim 4 .  
     
     
         12 . A cell derived from the P-PHSC of  claim 5 .  
     
     
         13 . A cell derived from the P-PHSC of  claim 6 .  
     
     
         14 . A cell derived from the P-PHSC of  claim 7 .  
     
     
         15 . A method of potentiating transduction of P-PHSC with an rAAV-vector comprising: 
 harvesting P-PHSC;    after said harvesting, culturing said harvested P-PHSC in a culture medium allowing for proliferation of said P-PHSC, said culture medium comprising a hemopoietic growth factor; and    after said culturing, introducing an rAAV-vector into said cultured P-PHSC to genetically modify said cultured P-PHSC.    
     
     
         16 . The method of  claim 15  wherein said hemopoietic growth factor comprises interleukin-3 or a functional analog or fragment thereof.