THERAPEUTIC RETROVIRAL VECTORS FOR GENE THERAPY
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.