Chicken HS4 Insulators Have Minimal Functions in Human Hematopoietic Cells That Were Transduced with An HIV1-Based Lentiviral Vector.
Authors: Uchida Naoya , Washington Kareem , Hsieh Matthew M. , Tisdale John F. 
Published: October 2019

Abstract 3570

Poster Board III-507

For the hemoglobin disorders, hematopoietic stem cell gene transfer is potentially curative, yet this strategy requires high-level β-globin gene expression among erythroid cells. Position effects, which are imparted by chromosomal position and chromatin structure, induce clonal variability of transgene expression. Recent work demonstrates that the chicken HS4 insulator element reduces position effects, resulting in consistently high-level expression of a therapeutic β-globin gene in the MEL cell line. In this study, we evaluated the effects of HS4 insulators on lentiviral vector titers and transgene expression among transduced human hematopoietic cells.

We constructed various types of insulated lentiviral vectors using a reverse oriented GFP under the control of the MSCV-LTR promoter (rMpGFP) or a conventional reverse oriented β-globin expression cassette, in which the globin gene was changed to GFP (BGpGFP). A full-length HS4 insulator (1.2 kb HS4), tandem HS4 core insulator (2 × 250 b HS4), and a single core insulator (250 b HS4) were inserted into the 3′ LTR. The insulator elements were inserted in both forward (F) and reverse (R) orientations.

Vector titers were significantly decreased by insertion of the 1.2 kb HS4 and 2 × 250 b HS4 in both orientations and both vector constructs, compared to uninsulated vectors (p<0.05), with the degree dependent on fragment size. Interestingly, reverse-oriented insulators showed better vector titers when compared to forward-oriented insulators for all types of insulator fragments except the 2 × 250 b HS4 in rMpGFP vectors (p<0.05).

We next evaluated GFP expression from various insulated rMpGFP vectors in GPA+ human erythroid cells that originated from transduced CD34+ cells (MOI=3) (Figure). The %GFP was decreased by 1.2 kb HS4 and 2 × 250 b HS4 insulators in both orientations, compared to the uninsulated vector (p<0.05). All insulated vector constructs had a tendency to lower CVs, there was no significant difference except for the 1.2 kb HS4 F vector (p<0.05). There was no significant difference of MFIs between all types of insulated and uninsulated vectors. In order to evaluate insulator function for the BGpGFP vectors in human hematopoietic cells, we practically chose the 250 b HS4 R because it did not decrease vector titers and the 1.2 kb HS4 showed 5-fold lower transduction efficiency in human erythroid cells. During erythorid culture of transduced human erythroid cells, %GFP and MFIs decreased whereas CVs increased,showing chromosomal position effects. The 250 b HS4 R insulator showed lower %GFP and lower MFIs (MOI=20) (p<0.05 on day 13 and 20), compared to those of the uninsulated vector. There was no significant difference in CVs. After MOI escalation of BGpGFP vectors (day13), the insulated vector showed lower %GFP at MOI 10, 25, and 50 (p<0.05) and lower overall GFP expression (%GFP x MFI) at MOI 25 and 50 (p<0.05) compared to uninsulated vector. These data demonstrated that inclusion of HS4 insulator elements decreases GFP expression, which is not overcome by increasing MOI. We then performed transduced hematopoietic stem cell transplantation in a human xenograft mouse model using a 250 b HS4 R insulated rMpGFP vector. In the human CD45+ fraction of mouse peripheral blood cells, the insulator element decreased both %GFP and MFIs at 4 and 8 weeks after transplantation (p<0.05). There was no significant difference of CVs among the insulated and uninsulated vector at all time points.

These data demonstrate that the inclusion of HS4 insulator elements lowers viral titers, reduces efficiency of transduction and produces minimal effects on transgene expression among human hematopoietic cells in vitro and in vivo

Disclosures:

No relevant conflicts of interest to declare.