Elements of a Chromatin Insulator Necessary for Consistent, High-Level Globin Gene Expression
Authors: Arumugam Paritha I. , Xia Ping , Urbinati Fabrizia , Malik Punam 
Published: April 2020

Vectors flanked by the chicken b-globin locus hypersensitive site-4 (cHS4) insulator provide consistent, improved expression (chromatin barrier activity) and reduced genotoxicity (enhancer-blocking activity); features important for randomly integrating viral vectors. However, full-length 1.2Kb cHS4 reduces vector titers significantly. Initial studies mapped the enhancer blocking activity of cHS4 to the first 250bp ‘core’ in a copy-number dependent manner (Felsenfeld and colleagues). However, elements required for the barrier effect are not well defined. We studied various regions of cHS4 for chromatin barrier effect. Beta-globin-LCR lentivirus vectors were constructed with the core (sBGC), 2 copies of the core (sBG2C), 400bp (sBG400) and 800bp (sBG800) fragments (both including the core) and compared to the un-insulated vector (sBG) and the vector with the full-length cHS4 (sBG-I). Barrier activity (improved probability and consistency of expression from individual integrants) in MEL cells and the clonal progeny of hematopoietic stem cells was analyzed. Single-copy MEL clones showed that sBG-I clones had a significantly higher proportion of hβ+ cells/clone. However, proportion of hβ+ cells from all other vectors carrying various fragments of cHS4 were not significantly different from the un-insulated vector (Table 1). Interestingly, all clones carrying a fragment containing the core showed reduced clonal variegation (coefficient of variation of hβ expression). We next analyzed hβ expression from the sBG, sBGC, sBG2C, sBG400 and sBG-I vectors in primary and secondary thalassemia (Hbb th3/+) mice. In the primary mice and secondary CFU-S analyzed thus far (Table 2), it appears that all vectors with cHS4 insulator fragments containing the core provide some barrier activity, but the full-length insulator is necessary for maximum barrier effect. To corroborate whether elements outside the core contribute to barrier activity, we also tested vectors with inert DNA spacers in addition to the core (sBG400−S, sBG-800−S and sBG1200−S). Single copy MEL clones confirmed that the sBG400−S, sBG800−S and 1200−S vectors had significantly lower proportion of hβ+ cells/clone [41±4.4%, 41±7% and 28±6% respectively, n=36]. These data suggest that regions of 950 bp region of cHS4 outside the core contains elements that cooperate with the core for optimal barrier activity. These studies have important implications for vector design for gene therapy for thalassemia, where variability in expression from uninsulated vectors compromizes efficacy.

Table 1. β globin expression in MEL cell clones from vectors carrying portions of the cHS4 insulator sBG sBG c sBG 2c sBG 400 sBG 800 sBG-I Values are expressed as Mean±SEM, *P&lt;0.05, **P&lt;0.01 by ANOVA (Dunnet: comparisons of all vectors to the control vector, sBG) % hβ+ cells 59±5 49±6 51±3 51±3 49±6 84±3** CV 53±4 37±3** 40±2** 42±1.5** 42±3** 36±2** Table 2. β globin expression in primary thalassemia mice and in the clonal progeny of hematopoietic stem cells of secondary mice Primary mice (16 weeks) Mock sBG sBG c sBG 2c sBG 400 sBG-I Values are expressed as Mean ± SEM, *P&lt;0.05, **P&lt;0.01 by ANOVA (Dunnett: comparison of all vectors to the control, sBG) HCT (%) 20±3 32±3 36±2 37±2 37±3 39±2 Reticulocytes (%) 28±2 16±5 11±1** 13±6** 7±2** 7±2** %HbA (mαhβ by HPLC)/vector copy NA 32±19 24±5 29±3 31±3 43±4* % hβ+RBC/ vector copy NA 36±11 57±8* 67±13* 76±17* 104±18**     Secondary CFU-S (Single Copy) % hβ+ cells/clone NA 26±3 45±3* 34±2 48±9* 75±3** CV NA 88±9 68±2* 71±2* 72±3 61±2**