Gene therapy methods
The present invention generally provides improved gene therapy vectors, cell-based compositions, and methods of using the same in methods of gene therapy. The present invention further provides improved gene therapy compositions for expanding hematopoietic cells and related methods for treatment of diseases, disorders, and conditions of the hematopoietic system such as thalassemias and anemias.
1. A vector comprising:
a) a left (5′) retroviral LTR;
b) a hematopoietic cell expression control sequence that comprises an erythroid cell specific promoter and optionally an erythroid cell specific enhancer, operably linked to a globin gene;
c) an ubiquitous expression control sequence operably linked to a truncated erythropoietin receptor (tEpoR), wherein the tEpoR comprises a C-terminal truncation that reduces the turnover of the tEpoR compared to an endogenous erythropoietin receptor (EpoR) or that increases the half-life of the tEpoR compared to an endogenous erythropoietin receptor (EpoR); and
d) a right (3′) retroviral LTR.
2. The vector of claim 1 , wherein the hematopoietic cell expression control sequence is selected from the group consisting of: a human β-globin promoter; a human β-globin LCR; and a human α-globin HS40 enhancer and an ankyrin-1 promoter.
3. The vector of claim 1 , wherein:
(a) the globin gene is selected from the group consisting of: human β-globin, human δ-globin, and human γ-globin; or
(b) the globin gene is selected from the group consisting of: a human β A -globin gene encoding a threonine to glutamine mutation at codon 87 (β A-T87Q ) or a human β A -globin gene.
4. The vector of claim 1 , wherein the tEpoR comprises a C-terminal truncation.
5. The vector of claim 4 , wherein the tEpoR comprises
(a) a C-terminal truncation of 80 to 90 amino acids;
(b) a C-terminal truncation of 85 to 95 amino acids; or
(c) a C-terminal truncation of about 83, or about 91 amino acids.
6. The vector of claim 1 , wherein:
(a) the vector is a lentivirus vector;
(b) the 5′ LTR or 3′ LTR is a lentivirus LTR;
(c) the 5′ LTR and 3′ LTR are lentivirus LTRs;
(d) the promoter of the 5′ LTR is replaced with a heterologous promoter;
(e) the 3′ LTR comprises one or more modifications;
(f) the 3′ LTR comprises one or more deletions; or
(g) the 3′ LTR is a self-inactivating (SIN) LTR.
7. The vector of claim 1 , further comprising one or more of a Psi packaging sequence (Ψ + ), a central polypurine tract/DNA flap (cPPT/FLAP), a retroviral export element, a posttranscriptional regulatory element, an insulator element, a polyadenylation sequence, a selectable marker, and a cell suicide gene.
8. A composition comprising a pharmaceutically acceptable carrier and the vector of claim 1 .
9. A human cell comprising the vector of claim 1 .
10. The cell of claim 9 , wherein:
(a) the cell is selected from the group consisting of: an embryonic stem cell, an adult stem cell, an adult progenitor cell, and a differentiated adult cell;
(b) the cell is a hematopoietic stem cell or a hematopoietic progenitor cell;
(c) the cell is a hematopoietic stem cell or a hematopoietic progenitor cell and the source of the stem or progenitor cell is bone marrow, cord blood, placental blood, or peripheral blood; or
(d) the cell is transduced with the vector.
11. A composition comprising a pharmaceutically acceptable carrier and the cell of claim 9 .
12. A composition comprising a pharmaceutically acceptable carrier and the cell of claim 10 .