Viral Vectors for Prophylaxis and Therapy of Hemoglobinopathies

Abstract

Provided are compositions and methods for inducing expression of human beta-globin in erythrocytes for use in prophylaxis and/or therapy of a hemoglobinopathy in an individual. The method generally entails introducing into CD34+ cells a polynucleotide encoding: i) a 5′ long terminal repeat (LTR) and a self-inactivating 3′ LTR; ii) at least one polyadenylation signal; iii) at least one promoter; iv) a globin gene locus control region (LCR); v) an ankyrin insulator element (Ank); vi) a Woodchuck Post-Regulatory Element (WPRE) configured such that the WPRE does not integrate into a target genome; and vii) a sequence that is a reverse complement of a sequence encoding human beta-globin, and can include beta-globin that has a βT87Q mutation. Intron 2 of the beta globin gene can be a complete intron. Modified erythrocyte progenitor cells, recombinant vectors and virions comprising recombinant polynucleotides, and methods of making the vectors and virions are included.

What is claimed is: 
     
         1 . A method for inducing expression of human beta-globin in erythrocytes for use in prophylaxis and/or therapy of a hemoglobinopathy in an individual comprising introducing into erythrocyte progenitor cells a polynucleotide encoding:
 i) a 5′ long terminal repeat (LTR) and a self-inactivating 3′ LTR;   ii) at least one polyadenylation signal;   iii) at least one promoter;   iv) a globin gene locus control region (LCR);   v) an ankyrin insulator element (Ank);   vi) a Woodchuck Post-Regulatory Element (WPRE) configured such that the WPRE does not integrate into a target genome; and   vii) a sequence that is a reverse complement of a sequence encoding modified human beta-globin comprising a (3T87Q mutation (B-globinM), wherein the sequence encoding the B-globinM comprises a first intron (intron 1) between exon 1 and exon 2, and a second intron (intron 2) between exon 2 and exon 3 of said B-globinM sequence, wherein intron 2 comprises more than 476 nucleotides of human B-globinM intron 2 sequence;   wherein, subsequent to the introducing, the erythrocyte progenitor cells differentiate into erythrocytes, wherein the erythrocytes are present in the individual and the erythrocytes produce more human beta-globin than a control.   
     
     
         2 . The method of  claim 1 , wherein the control comprises a human beta-globin value obtained from control cells, wherein the control cells comprise erythrocytes from an individual who has the hemoglobinopathy, wherein the erythrocytes are progeny of cells into which a control viral vector is introduced, the control viral vector comprising the 5′ LTR, the 3′ LTR, the at least one polyadenylation signal, the at least one promoter, the LCR, the Ank, a WPRE, and the sequence encoding the B-globinM, but wherein the sequence encoding the B-globinM in the control viral vector comprises 476 or fewer nucleotides of the human B-globinM intron 2 sequence. 
     
     
         3 . The method of  claim 1 , wherein the lentiviral vector further comprise a sequence encoding a fusion of an Ldb1 transcription factor and a zinc finger (ZF) domain. 
     
     
         4 . The method of  claim 1 , wherein the lentiviral vector further comprises a sequence encoding an RNA polynucleotide that is a reverse complement of mRNA encoding transferrin receptor 1, and wherein the RNA polynucleotide comprises a microRNA or an shRNA sequence and is capable of decreasing transferrin receptor 1 mRNA in an RNAi-mediated process. 
     
     
         5 . The method of  claim 1 , wherein the erythrocyte progenitor cells comprise CD34+ cells, wherein the CD34+ cells are separated from the individual before the introducing the lentiviral vector to the cells, and wherein the CD34+ cells are introduced into the individual subsequent to the introducing of the lentiviral vector. 
     
     
         6 . The method of  claim 1 , wherein the intron 2 comprises 851 nucleotides of the adult human B-globinM intron 2 sequence. 
     
     
         7 . The method of  claim 1 , wherein the increased human beta-globin comprises adult hemoglobin, fetal hemoglobin, B-globinM, or a combination thereof. 
     
     
         8 . The method of  claim 7 , wherein the hemoglobinopathy comprises sickle-cell anemia (SCA) or beta-thalassemia. 
     
     
         9 . A lentiviral vector for inducing expression of human beta-globin in erythrocytes and/or erythrocyte progenitor cells for use in prophylaxis and/or therapy of a hemoglobinopathy in an individual, the lentiviral vector comprising:
 i) a 5′ long terminal repeat (LTR) and a self-inactivating 3′ LTR;   ii) at least one polyadenylation signal;   iii) at least one promoter;   iv) a globin gene locus control region (LCR);   v) an ankyrin insulator element (Ank);   vi) a Woodchuck Post-Regulatory Element (WPRE) configured such that the WPRE does not integrate into a target genome;   vii) a sequence that is a reverse complement of a sequence encoding modified adult human beta-globin comprising a βT87Q mutation (B-globinM), wherein the sequence encoding the B-globinM comprises a first intron (intron 1) between exon 1 and exon 2, and a second intron (intron 2) between exon 2 and exon 3 of said B-globinM sequence, wherein intron 2 comprises more than 476 nucleotides of adult human B-globinM intron 2 sequence.   
     
     
         10 . The lentiviral vector of  claim 9 , wherein the lentiviral vector further comprise a sequence encoding a fusion of an Ldb1 transcription factor and a zinc finger (ZF) domain. 
     
     
         11 . The lentiviral vector of  claim 9 , wherein the lentiviral vector further comprises a sequence encoding a RNA polynucleotide that a reverse complement of mRNA encoding transferrin receptor 1, and wherein the RNA polynucleotide comprises a microRNA or a shRNA sequence and is capable of decreasing transferrin receptor 1 mRNA in a RNAi-mediated process. 
     
     
         12 . The lentiviral vector  claim 9 , wherein the lentiviral vector is present in CD34+ cells, wherein the CD34+ cells have been separated from an individual who has a hemoglobinopathy. 
     
     
         13 . The lentiviral vector of  claim 12 , wherein the lentiviral vector is present in a virion. 
     
     
         14 . A pharmaceutical composition comprising viral particles, wherein the viral particles each comprise a RNA strand which comprises a lentiviral vector of  claim 9 . 
     
     
         15 . A method of making a viral particle preparation for use in prophylaxis and/or therapy for one or more hemoglobinopathies comprising introducing a plasmid encoding a lentiviral vector of  claim 1  into packaging cells which comprise a DNA packaging plasmid which encodes at least one virion protein, and a DNA envelope plasmid which encodes an envelope protein, and allowing expression of the virion protein and the envelope protein such that viral particles form, and separating the viral particles from the packaging cells.