Optimized lentiviral vector compromising minimal enhancer elements for stem cell gene therapy of hemoglobinopathies
In certain embodiments a lentiviral vector having optimized reduced size LCR with improved enhancer activity is provided. In certain embodiments direct treatment of a subject by direct introduction of the vector(s) described herein is contemplated. The lentiviral compositions may be formulated for delivery by any available route including, but not limited to parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, rectal, and vaginal.
1 . A recombinant lentiviral vector (LV) comprising: an expression cassette comprising a nucleic acid construct comprising:
(a) a human β-globin locus control region (LCR) sequence selected from the group consisting of:
(i) a sequence consisting of reduced length HS sequences set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10;
(ii) a sequence consisting of reduced length HS sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20;
(iii) a sequence consisting of reduced length HS sequences set forth in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29;
(iv) a sequence consisting of reduced length HS sequences set forth in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36; or
(v) a sequence consisting of reduced length HS sequences set forth in SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42; and
(b) a heterologous gene to be expressed by said construct operably linked to said human β-globin LCR sequence; and
wherein said LV is a TAT-independent and self-inactivating (SIN) lentiviral vector.
2 . The vector of claim 1 , wherein said human β-globin locus control region (LCR consists of the reduced length HS sequences consisting of the sequences set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
3 . The vector of claim 1 , wherein said human β-globin locus control region (LCR consists of the reduced length HS sequences consisting of the sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
4 . The vector of claim 1 , wherein said human β-globin locus control region (LCR consists of the reduced length HS sequences consisting of the sequences set forth in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29.
5 . The vector of claim 1 , wherein said human β-globin locus control region (LCR consists of the reduced length HS sequences consisting of the sequences set forth in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36.
6 . The vector of claim 1 , wherein said human β-globin locus control region (LCR consists of the reduced length HS sequences consisting of the sequences set forth in SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42.
7 . The vector of claim 1 , wherein:
said heterologous gene comprises a recombinant human β-globin gene encoding a β-globin polypeptide; or
said heterologous gene comprises a wild-type β-globin gene; or
said heterologous gene comprises a recombinant human β-globin gene that comprises an anti-sickling human β-globin gene encoding an anti-sickling β-globin polypeptide.
8 . The vector of claim 7 , wherein said anti-sickling human β-globin gene encoding an anti-sickling β-globin polypeptide comprises one or more mutations selected from the group consisting of Gly16Asp, Glu22Ala, and Thr87Gln.
9 . The vector of claim 1 , wherein said
vector comprises a human Ankyrin insulator element; and/or
said vector comprises a w region vector genome packaging signal; and/or
said vector comprises a 5′ LTR that comprises a CMV enhancer/promoter; and/or
said vector comprises a Rev Responsive Element (RRE); and/or
said vector comprises a central polypurine tract; and/or
said vector comprises a post-translational regulatory element.
10 . The vector of claim 1 , wherein: said vector comprises the nucleic acid sequence set forth in SEQ ID NO: 1; or said vector comprises the nucleic acid sequence set forth in SEQ ID NO: 11; or said vector comprises the nucleic acid sequence set forth in SEQ ID NO: 21; or said vector comprises the nucleic acid sequence set forth in SEQ ID NO: 30; or said vector comprises the nucleic acid sequence set forth in SEQ ID NO: 37.
11 . A host cell transduced with a vector of claim 1 .
12 . The host cell of claim 11 , wherein the cell is a stem cell.
13 . The host cell of claim 12 , wherein said cell is a stem cell derived from bone marrow, or from umbilical cord blood, or from peripheral blood.
14 . The host cell of claim 11 , wherein the cell is a human hematopoietic progenitor cell.
15 . A method of treating a hemoglobinopathy in a subject, said method comprising:
transducing a stem cell and/or progenitor cell from said subject with a vector of claim 1 , wherein said heterologous gene comprises an anti-sickling human β-globin gene encoding an anti-sickling β-globin polypeptide; and
transplanting said transduced cell or cells derived therefrom into said subject, wherein said cells or derivatives therefrom express said anti-sickling human β-globin polypeptide.
16 . The method of claim 15 , wherein said hemoglobinopathy is sickle cell disease or β-thalassemia.