Therapeutic retroviral vectors for gene therapy

Abstract

Retroviral gene therapy vectors that are optimized for erythroid specific expression and treatment of hemoglobinopathic conditions are disclosed.

1 - 31 . (canceled) 
     
     
         32 . A self-inactivating (SIN) lentiviral vector comprising:
 a) a 5′ long terminal repeat (LTR);   b) an RNA export element;   c) a β-globin promoter;   d) a β-globin locus control region (LCR); and   e) a modified 3′ LTR comprising:
 i) at least one insulator element; or 
 ii) a poly (A) sequence; 
   wherein the β-globin promoter and β-globin LCR are operatively linked to a gene of interest.   
     
     
         33 . The vector of  claim 32 , wherein the 5′ LTR comprises a deletion compared to the wild-type 5′ LTR and a heterologous promoter. 
     
     
         34 . The vector of  claim 33 , wherein the heterologous promoter is a cytomegalovirus (CMV) promoter. 
     
     
         35 . The vector of  claim 32 , wherein the RNA export element comprises a hepatitis B virus post-transcriptional regulatory element (PRE) or a human immunodeficiency virus (HIV) rev response element (RRE). 
     
     
         36 . The vector of  claim 32 , comprising a lentiviral central polypurine tract or DNA FLAP (cPPT/FLAP). 
     
     
         37 . The vector of  claim 32 , wherein the β-globin LCR comprises DNase I hypersensitive sites 2, 3, and 4 from the human β-globin LCR. 
     
     
         38 . The vector of  claim 32 , comprising a human β-globin 3′ enhancer element. 
     
     
         39 . The vector of  claim 32 , comprising the at least one insulator element and the poly (A) sequence. 
     
     
         40 . The vector of  claim 32 , wherein the modified 3′ LTR comprises two insulator elements. 
     
     
         41 . The vector of  claim 32  or  claim 40 , comprising an insulator sequence as set forth in SEQ ID NO: 2. 
     
     
         42 . The vector of  claim 32  or  claim 40 , comprising an insulator sequence as set forth in nucleotides 8-49 of SEQ ID NO: 2. 
     
     
         43 . The vector of  claim 32 , wherein the lentivirus is selected from the group consisting of: human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), and simian immunodeficiency virus (SIV). 
     
     
         44 . The vector of  claim 32 , wherein the modified 3′ LTR comprises at least one deletion compared to the wild-type 3′ LTR. 
     
     
         45 . The vector of  claim 44 , comprising the at least one insulator element and the poly (A) sequence. 
     
     
         46 . The vector of  claim 45 , wherein the modified 3′ LTR comprises two insulator elements. 
     
     
         47 . The vector of any one of  claims 44 - 46 , comprising an insulator sequence set forth in SEQ ID NO: 2. 
     
     
         48 . The vector of any one of  claims 44 - 46 , comprising an insulator sequence as set forth in nucleotides 8-49 of SEQ ID NO: 2. 
     
     
         49 . The vector of  claim 32 , wherein the gene of interest encodes an antisickling protein or a globin gene. 
     
     
         50 . The vector of  claim 32 , wherein the gene of interest encodes a human β-globin gene, a human δ-globin gene, or a human β A-T87Q -globin gene. 
     
     
         51 . The vector of  claim 32 , comprising a nucleic acid cassette comprising a suicide gene operably linked to a promoter or a gene for in vivo selection of the cell. 
     
     
         52 . The vector of  claim 51 , wherein the suicide gene is HSV thymidine kinase (HSV-Tk). 
     
     
         53 . The vector of  claim 51 , wherein the gene for in vivo selection is methylguanine methyltransferase (MGMT). 
     
     
         54 . A cell transduced with the vector of  claim 32 . 
     
     
         55 . The transduced cell of  claim 54 , wherein the cell is an embryonic stem cell, a somatic stem cell, or a progenitor cell. 
     
     
         56 . The transduced cell of  claim 55 , wherein the cell is a bone marrow cell, a hematopoietic stem cell, or a hematopoietic progenitor cell. 
     
     
         57 . The transduced cell of  claim 55 , wherein the cell is an erythrocyte. 
     
     
         58 . A method of transplanting transduced cells to a subject having a hemoglobinopathy comprising administering the transduced cells of  claim 54  to the subject. 
     
     
         59 . The method of  claim 58 , wherein the transduced cells express a therapeutically effective amount of a human β-globin gene, a human δ-globin gene, or a human β A-T87Q -globin gene in the subject. 
     
     
         60 . The method of  claim 58 , wherein the hemoglobinopathy is selected from the group consisting of: hemoglobin sickle cell disease (SCD), sickle cell anemia, and β-thalassemia.