Evaluation of a γ-Globin Lentiviral Vector in Sickle Cell Mice and Pigtail Macaques

Abstract Increased levels of red cell fetal hemogloblin (α2 γ2; HbF), whether due to hereditary persistence of HbF or from induction with hydroxyurea therapy, effectively ameliorate sickle cell disease (SCD). Therefore, we developed an erythroid-specific, γ-globin lentiviral vector for hematopoietic stem cell (HSC)-targeted gene therapy with the goal of permanent, high level expression of HbF in sickle red cells. The vector contained the γ-globin gene driven by 3.1 kb of transcriptional regulatory sequences from the β-globin LCR and a 130 bp β-globin promoter. Since adult erythroid cells have β-globin mRNA 3′UTR binding proteins that enhance β-globin mRNA stability, we replaced the native γ-globin 3′UTR with its β-globin counterpart. We tested the therapeutic efficacy of this vector using the BERK sickle cell mouse model. Five months following transplant, mice that received transduced lineage-depleted sickle steady-state bone marrow (BM) cells (n=10) expressed the g-globin transgene in 95% ± 2% of RBCs. We observed levels of HbF that equaled that of the endogenous HbS (HbF 48% ± 3% of total Hb). This was achieved with an average BM vector copy number of 1.7 ± 0.2 and led to correction of both the severe anemia and end-organ damage characterizing this SCD strain. Globin vector mice had a Hb level of 12.2 ± 0.2 g/dL, compared to 7.1 ± 0.3 g/dL of mice (n=16) transplanted with cells transduced with a control GFP vector. Urine concentrating ability was normal in globin vector mice, while severely impaired in control mice. At necropsy, minimal evidence of sickle-related organ damage was found in the globin vector recipient group. In contrast, severe renal, hepatic, splenic and pulmonary pathology was observed in control, mock-transduced animals. We then transplanted the BM from 6 primary recipients of globin vector-transduced cells into 23 secondary recipients. Five months after transplant, these animals maintained HbF levels similar to those of their primary donors, along with persistent resolution of anemia. This suggested that HSCs were transduced and that vector silencing was minimal. We then evaluated this vector using non-human primate CD34+ cells. Steady-state BM CD34+ cells from several different pigtail macaques were transduced with the globin lentiviral vector or with a GFP control vector. The GFP vector achieved an average transduction rate of 57% ± 6% (n=6) into CD34+ cells and 76% ± 9% into CFU, as judged by GFP expression. Similar high levels of gene transfer were obtained with the globin vector. Bulk CD34+ cells transduced with the globin vector and then cultured for 5 days demonstrated an average vector copy number of 0.6–1.0 as judged by Southern blot analysis and qPCR. High level transduction of CFU was also obtained as 12/16 and 16/16 colonies in two separate experiments were positive for the globin vector by PCR analysis of colony DNA. We are in the process of comparing globin gene transfer and expression with that of our standard GFP vector in the pigtail macaque autologous transplant model by transplanting a graft consisting of 50% globin lentiviral vector-transduced CD34+ cells and 50% GFP lentiviral vector-transduced cells.