Use of a Lentiviral Vector Encoding γ-Globin and the MGMT Drug Resistance Gene Coupled with In Vivo Drug Selection To Cure β-Thalassemia.
Authors: Zhao Huifen , Pestina Tamara I. , Hargrove Phillip W. , Persons Derek A. 
Published: April 2020

Abstract
Since increased levels of fetal hemoglobin can significantly ameliorate the severity of β-thalassemia, a strategy using autologous, stem cell-targeted transfer of a γ-globin gene capable of being expressed at high levels in adult erythroid cells may offer a potential cure. Previously, we and other groups have demonstrated correction of a murine model of severe β-thalassemia with an average globin lentiviral vector copy number of ∼1, meaning most, if not all, hematopoietic stem cells (HSCs) were genetically modified with the vector genome. Although mouse HSCs are easily modified with lentiviral vectors, evidence from non-human primate studies predicts much lower levels of lentiviral gene transfer into human HSCs. Therefore, we incorporated a drug resistance gene, methylguanine methyltransferase (MGMT), into our globin vector to allow in vivo selection of genetically modified cells following transplantation through the administration of drugs that kill the disease-causing, non-modified HSCs. We utilized a 3rd generation, self-inactivating vector design (SJ1) containing the γ-globin gene in reverse orientation under the transcriptional control of 3.1 kb of elements from the β-globin locus control region (LCR) coupled to a 130 bp minimal β-globin promoter. The MGMT cDNA was positioned in the opposite orientation from the globin cassette and was driven by an internal MSCV U3 promoter. Remarkably, this complex vector was produced at a mean unconcentrated titer of 7 × 105 units/ml; clinically relevant titers of &gt;108 per ml were obtained by ultracentrifugation. We transplanted lethally irradiated mice with lineage negative β-thalassemic bone marrow cells transduced with the γ-globin/MGMT vector. Twelve weeks following transplant, animals were divided into 2 groups: a control group (CON; n=6) that received no further manipulation and was only observed, and a treatment group (RX; n=7) that received BCNU and benzylguanine every 6–8 weeks for 3 courses. The baseline percentage of F cells was similarly low in both groups (CON 4.6% ± 2.1 vs. RX 11.3% ± 4.3, p= 0.21) and total levels of HbF in the blood were virtually undetectable (&lt; 5%). All animals were anemic with similar Hb values in both groups (CON 9.4 g/dL ± 0.3 vs. RX group 9.8 ± 0.3, p= 0.31) and blood smears showed typical RBC morphologic features of β-thalassemia. Although one CON mouse died during the experiment, RX mice tolerated the treatment well with no deaths. One month after the final BCNU/BG treatment, all mice were re-analyzed for their hematologic parameters. Five of 7 mice in the RX group demonstrated a significant increase in F cells (mean of 11% rising to 46%) and, importantly, total blood levels of HbF increased from a mean of less than 5% to 21% (range 13–30%). These increased levels of HbF resulted in resolution of anemia in the responding animals (Hb of 9.8 g/dL rising to 11.1). Blood smears of these animals showed normalization of RBC morphology. In contrast, the CON group of mice showed no improvement in any of these parameters compared to baseline and remained anemic (Hb 9.6). Secondary transplantation and clonal analysis studies of hematopoiesis in the responding RX mice is ongoing and will address both the durability of cure and the diversity of the stem cell population after vivo selection. Analysis of the effects of selection on the mean globin expression per cell will be presented. These data are the first to demonstrate the usefulness of in vivo selection drug selection in β-thalassemia using a globin lentiviral vector.