Self-Inactivating Lentiviral Vectors Flanked by a Chromatin Insulator Element Result in Increased, Consistent Expression of Human Beta Globin.
Authors: Arumugam Paritha I. , Scholes Jessica , Perelman Natalya , Zarzuela Alexander , Malik Punam 
Published: October 2019

Lentiviral vectors carrying the human β-globin gene (hβ) and hypersensitive sites 2, 3 and 4 of the hβ locus control region (LCR) have revolutionized the field of gene therapy for hemoglobinopathies, resulting in correction in mouse and human models of β-thalassemia. However, their random integration into host chromosome results in highly variable hβ expression, dependent on the flanking host chromatin (chromosomal position effects). Moreover, the recent occurrence of leukemogenesis from activation of a cellular oncogene by the viral enhancer elements calls for safer vector designs, with expression cassettes that can be ‘insulated’ from flanking cellular genes. We analyzed the role of the chicken β-globin locus hypersensitive site 4 insulator element (cHS4) in a self-inactivating (SIN) lentiviral vector. The BGM vector carrying the hβ/LCR was compared to an analogous vector BGMI, where the cHS4 was inserted in the SIN deletion to flank the hβ/LCR at both ends upon integration. Both vectors additionally carried the methyl guanine methyl transferase (MGMT) cDNA to enrich for genetically modified cells. First, murine erythroleukemia (MEL) cells were transduced at <5% transduction efficiency in three separate experiments to generate single copy clones, transduced clones identified by PCR (‘Unselected’ clones) and analyzed for hβ protein by FACS and mRNA by RNase Protection Assay (RPA). ‘Unselected’ BGMI+-MEL clones had a higher proportion of hβ+ cells (68±3%) compared to BGM+ clones (36±9%, n=24; p<0.001). Additionally, the coefficient of variation of hβ expression (CV) in each clone was reduced in BGMI+ clones: 168±20 vs. 327±64 in BGM (P<0.02). RPA showed a 2-fold increase in hβ/total mα-globin mRNA with the BGMI vector [BGMI 45±6% vs. BGM 21±7%, n=24, p<0.01]. Next, the same pool of MEL cells transduced with BGM and BGMI at <5% transduction efficiency were selected with BG/BCNU and 86 single-copy clones isolated (‘Selected’ clones). ‘Selected’ BGMI+-MEL clones also had a higher proportion of hβ+ cells (BGMI 80±15%, n=54 vs. BGM 72±20%, n=32; p<0.03), with reduced CV (140±2.8 vs. 170±13, P<0.01) and higher hβ-mRNA [BGMI+ 83.6%± 40 vs. 49.6%±25 in BGM+ clones (n=24, p<0.02)]. In vivo studies confirmed MEL cell results: lethally irradiated normal mice were transplanted with BGM and BGMI-transduced thalassemia hematopoietic stem cells (MOI 20). Engraftment and vector copy number in both groups were similar (66±15% vs. 68±10%, and 0.21 vs 0.17 in BGMI and BGM groups, respectively, n=12). While, 18±4% of murine RBC expressed hβ in the BGMI group, only 4±1.5% expressed hβ in the BGM group of mice by FACS (p<0.001). There was a 4-fold increase in chimeric hemoglobin (mα-hβ) in the BGMI mice (13% ± 4 vs. 3±2% in BGM mice) by hemoglobin electrophoresis (p<0.001). Secondary colony forming units-spleen (CFU-S) derived from these mice 12 weeks after transplant showed increased numbers of hβ+ cells in BGMI CFU-S (21±4% vs. 8±4% BGM-CFU-S, n=30, p<0.03) with reduced CV in BGMI CFU-S (698±91 vs. 987±99 in BGM CFU-S, p<0.04). MGMT selection and secondary transplants in BGM and BGMI mice are underway. Taken together, ‘insulated’ SIN-hβ/LCR lentiviral vectors increased the probability of expression of integrants and reduced chromatin position effects, resulting in consistent and higher hβ expression for gene therapy of β-thalassemia. The enhancer blocking effect of the cHS4, although not tested here, would further improve bio-safety of these lineage-specific, self-inactivated vectors.