67. The Chicken Hypersensitive Site (cHS4) Insulator Element Reduces Position Effects Thereby Increasing Expression from Globin Lentiviral Vectors
Authors: Paritha I. Arumugam, Jessica Scholes, Ping Xia, Natalya Perelman, Alexander Zarzuela, Jiing-Kuan Yee, Punam Malik
Journal: Molecular Therapy (2006)

Lentiviral vectors (LV) carrying the human β-globin gene (hβ) and locus control region (LCR) have changed the field of gene therapy for hemoglobinopathies. However, their random integration into host cells results in variable hβ expression from chromatin position effects. We analyzed the role of a 1.2 kb chicken β-globin locus hypersensitive site 4 insulator element (cHS4) in a self-inactivating (SIN) LV. The BGM vector (carrying hβ/LCR) was compared to an analogous vector BGMI, with cHS4 such that it flanks the provirus upon integration. Both vectors additionally carried the mutant P140K methyl guanine methyl transferase (MGMTP140K) cDNA to enrich for genetically modified cells. First, murine erythroleukemia (MEL) cells were transduced at <5% transduction efficiency to generate single-copy clones and transduced clones identified by PCR ('Unselected' clones) and analyzed for hβ expression by FACS and 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) and a reduced coefficient of variation (CV) of hβ expression: 168±20 vs. 327±64 in BGM (P<0.02). RPA showed a 2-fold increase in hβ/total mα-globin mRNA: BGMI 45±6% vs. BGM 21±7%, n=24, P<0.01. Next, MEL cells were selected with BG/BCNU and single-copy clones isolated ('Selected' clones). 'Selected' BGMI+-MEL clones also had a higher proportion of hβ+ cells compared to BGM+ clones (80±15% vs. 72±20%, n=86, 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, n=24, P<0.02). Two additional vectors termed BG and BGI (with cHS4) were constructed with the hβ/LCR but no MGMTP140K cassette. RPA showed that BGI+ MEL pools expressed 85% hβ/total mα-globin/ vector copy as compared to 38% from BG. BG and BGI single copy MEL clones showed similar results. In in vivo studies 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 RBC expressed hβ in the BGMI group, only 4%±1.5% expressed hβ in the BGM group (P<0.001). There was a 4-fold increase in chimeric hemoglobin (mα- hβ) in BGMI mice (13%±4% vs. 3%±2% in BGM mice, P<0.001). Secondary colony forming units-spleen (CFU-S) derived from these mice showed increased numbers of hβ+ cells in BGMI (21%±4% vs. 8%±4% BGM, n=30, P<0.03) with reduced CV (698±91 vs. 987±99 in BGM, P<0.04). Taken together, 'insulated' SIN-hβ/LCR LV increased the probability of expression of integrants and reduced chromatin position effects, resulting in consistent and higher expression of hβ. The enhancer blocking effect of the cHS4, although not tested here, would further improve the bio-safety of these lineage- specific, SIN LV.