Permanent and panerythroid correction of murine beta thalassemia by multiple lentiviral integration in hematopoietic stem cells

Abstract

Achieving long-term pancellular expression of a transferred gene at therapeutic level in a given hematopoietic lineage remains an important goal of gene therapy. Advances have recently been made in the genetic correction of the hemoglobinopathies by means of lentiviral vectors and large locus control region (LCR) derivatives. However, panerythroid beta globin gene expression has not yet been achieved in beta thalassemic mice because of incomplete transduction of the hematopoietic stem cell compartment and position effect variegation of proviruses integrated at a single copy per genome. Here, we report the permanent, panerythroid correction of severe beta thalassemia in mice, resulting from a homozygous deletion of the beta major globin gene, by transplantation of syngeneic bone marrow transduced with an HIV-1-derived [beta globin gene/LCR] lentiviral vector also containing the Rev responsive element and the central polypurine tract/DNA flap. The viral titers produced were high enough to achieve transduction of virtually all of the hematopoietic stem cells in the graft with an average of three integrated proviral copies per genome in all transplanted mice; the transduction was sustained for >7 months in both primary and secondary transplants, at which time approximately 95% of the red blood cells in all mice contained human beta globin contributing to 32 +/- 4% of all beta-like globin chains. Hematological parameters approached complete phenotypic correction, as assessed by hemoglobin levels and reticulocyte and red blood cell counts. All circulating red blood cells became and remained normocytic and normochromic, and their density was normalized. Free alpha globin chains were completely cleared from red blood cell membranes, splenomegaly abated, and iron deposit was almost eliminated in liver sections. These findings indicate that virtually complete transduction of the hematopoietic stem cell compartment can be achieved by high-titer lentiviral vectors and that position effect variegation can be mitigated by multiple events of proviral integration to yield balanced, panerythroid expression. These results provide a solid foundation for the initiation of human clinical trials in beta thalassemia patients.

Fig 1.

(A) Diagram of the human β globin (β^A) lentiviral vector. HIV LTR, HIV type-1 long terminal repeat; ψ+, packaging signal; cPPT, central polypurine tract/DNA flap; RRE, Rev-responsive element; E, exon; IVS, intervening sequence; βP, β globin promoter (from SnaBI to Cap site); HS, hypersensitive site; ppt, polypurine tract. The 3′ β globin enhancer (up to downstream AvrII site), the 372-bp IVS2 deletion (indicated by the triangle) and DNase I hypersensitive sites, HS2 (SmaI to XbaI), HS3 (SacI to PvuII) and HS4 (StuI to SpeI) of the LCR are shown. (B) Proportion of peripheral blood RBCs expressing human β globin assessed by FACS after staining the cells with an antibody specific for human β globin. RBCs were from THAL mice transplanted 7 months previously with bone marrow exposed to the lenti-β globin vector. The viral titer used in experiment 1 (E1), was ≈2 × 10⁸ infectious units/ml and in experiments 2 and 3 (E2, E3) was ≈1.5 × 10⁹ infectious units/ml. (C) Expression of human β globin in RBCs of reconstituted mice. (Upper Left) FACS analysis of RBCs from a representative recipient of lenti-GFP-virus-transduced THAL bone marrow cells. RBCs from an unmanipulated mouse were used as a negative control. (Upper Right and Lower Left) FACS analysis of RBCs from a representative recipient of lenti-β globin-virus-transduced THAL bone marrow cells at 2 and 7 months after transplantation. (Lower Right) FACS analysis of RBCs from one of the secondary recipients transplanted with bone marrow cells from a primary donor at 6 months after transplantation.

Fig 2.

Analysis of human β^A globin protein and membrane-associated α globin protein in RBC of THAL recipients of lenti-β globin-transduced THAL bone marrow cells. (A and B) Quantification of human β^A by HPLC. (A) Representative HPLC profile of peripheral blood from a recipient 4 months after transplant. (B) Amount of human β^A in individual corrected THAL recipients as measured by HPLC and expressed as percent of total β globin. (C) Isoelectric focusing of RBC lysates from transplanted primary mice showing the expected species of Hb with two murine α and two human β chains. Lanes 1–4, primary recipients; lanes 5 and 6, secondary recipients of lenti-β globin-transduced THAL bone marrow cells. (D) Urea-Triton polyacrylamide gradient gel of RBC membranes to detect membrane-associated α globin. Lanes 1–4, recipients of lenti-GFP transduced and lanes 5–8, recipients of lenti-β globin-transduced THAL bone marrow cells.

Fig 3.

Improvement of hematological parameters in THAL recipients of lenti-β globin-transduced THAL bone marrow cells. Results shown are the mean ± SD for untransplanted, control THAL mice (n = 5), normal unmanipulated B6 mice (n = 5), and THAL mice transplanted 6 months previously with lenti-β globin-transduced cells (n = 8) or control lenti-GFP-transduced cells (n = 6). Changes in all hematologic parameters seen in THAL recipients of lenti-β globin-transduced cells were highly significant in comparison with nontransplanted, or control (GFP)-transplanted THAL mice (*, P < 0.001). Values for the RBC number and reticulocyte count in these corrected mice reached levels within the normal range of control B6 mice (P = 0.6 and 0.1, respectively).

Fig 4.

Phenotypic changes in RBCs and decrease in hemosiderin accumulation in the spleen and liver of transplanted THAL mice. Blood smear of a THAL mouse transplanted with the control GFP (A) or [β globin gene/LCR] lentivirus (B)-transduced bone marrow cells 7 months before analysis. (C) Blood smear obtained 6 months after the transplantation of THAL mice with marrow from a primary mouse reconstituted for 2 months with [β globin gene/LCR] lentivirus-transduced cells. (D) Blood smear prepared from a normal, unmanipulated adult B6 mouse. (E, G, I, and F, H, J) Perls staining of spleen and liver. (E and F) The spleen and liver of a representative, unmanipulated THAL mouse; (G and H) spleen and liver of a [β globin gene/LCR] corrected THAL mouse killed 6 months after transplantation; and (I and J) spleen and liver of an unmanipulated control B6 mouse. Arrows shown in H indicate cells in which mild iron accumulation was observed. (K) Percoll-Larex continuous density gradient analysis of RBCs. Lanes 1–4, primary recipients; lanes 5 and 6, secondary recipients of [β globin gene/LCR] lentivirus-transduced THAL bone marrow cells. (L) Spleens from unmanipulated THAL and B6 mice, and THAL recipients of GFP or [β globin gene/LCR] lentivirus-transduced THAL bone marrow.