The human ankyrin 1 promoter insulator sustains gene expression in a β-globin lentiviral vector in hematopoietic stem cells

Zulema Romero, Beatriz Campo-Fernandez, Jennifer Wherley, Michael L Kaufman, Fabrizia Urbinati, Aaron R Cooper, Megan D Hoban, Kismet M Baldwin, Dianne Lumaquin, Xiaoyan Wang, Shantha Senadheera, Roger P Hollis, Donald B Kohn, Zulema Romero, Beatriz Campo-Fernandez, Jennifer Wherley, Michael L Kaufman, Fabrizia Urbinati, Aaron R Cooper, Megan D Hoban, Kismet M Baldwin, Dianne Lumaquin, Xiaoyan Wang, Shantha Senadheera, Roger P Hollis, Donald B Kohn

Abstract

Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the β-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human β-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term β-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies.

Figures

Figure 1
Figure 1
The proviral maps of the βAS3-globin vectors. The AS3 provirus has the βAS3-globin expression cassette including the human β-globin gene exons (arrow heads) with three amino acid substitutions to encode the βAS3-globin protein, introns and 3′ and 5′ flanking regions, the β-globin mini-LCR with hypersensitive sites 2–4, the mutated Woodchuck hepatitis virus post-transcriptional regulatory element (ΔWPRE) and the 3′SIN-LTR containing the FB insulator which is copied into the 5’LTR after reverse transcription. (a) AS3 LV, noninsulated version lacking the FB element; (b) CCL-βAS3-FB parental vector (FB LV); (c) Ank-R LV includes one copy of the Ank insulator in the reverse orientation; (d) Ank-FR LV with two copies of the Ank insulator element flanking the expression cassette, one in forward and one in reverse orientation; (e) Ank-RR LV with two copies of the Ank insulator flanking the expression cassette, both in reverse orientation (all of the Ank-insulated LV retain the FB element); and (f) a positive control vector that includes a full-length copy (1.2 kb) of the cHS4 insulator in the 3′LTR that is copied to 5’LTR during reverse transcription.
Figure 2
Figure 2
Consistent expression of the βAS3-globin cassette in MEL cell clones carrying one copy of the Ank insulator. (a) The percentage of cells expressing βAS3-globin transgene was analyzed by flow cytometry after in vitro erythroid differentiation 2 months post-transduction. Each point represents one single vector copy/cell clone (AS3, n = 15; FB, n = 10; Ank-R, n = 11; Ank-FR, n = 11; Ank-RR, n = 8; and cHS4, n = 12). Bars represent average values for each group. One-way ANOVA with pairwise comparisons was used to compare the percentage of cells expressing βAS3-globin transgene between groups. The significance threshold used for P-value was 0.05. (b) Geometric mean of βAS3-globin expression of the samples shown in (a), and at the same time point. Bars represent average values for each group. No significant increase in the level of expression of the βAS3-globin transgene was observed due to the addition of the Ank insulator (one-way ANOVA with multiple comparisons, P > 0.05). (c) Mean values of the percentage of cells expressing βAS3-globin transgene. The average and SE of the mean (SEM) of the percentage of cells expressing βAS3-globin transgene measured in each group of clones at 2, 3, and 4 months post-transduction are summarized. No significant differences were observed among groups over time (repeated-measure ANOVA). (d) Percentage of the CV (%CV) of βAS3-globin expressing MEL cell clones over time. The %CV at each time point was calculated as the ratio of the SD to the average percentage of expressing cells of the clones of each group at each time point.
Figure 3
Figure 3
Transgene expression in clonal progeny from murine HSC. Percentage of βAS3-globin mRNA normalized per VCN from CFU-S in secondary and tertiary recipients harvested 10 days post-transplant. Bars represent average values for each group and SD. The percentage of CV (%CV) shown at the bottom of the graph was calculated as the ratio of the SD to the mean of βAS3-globin levels per VCN from the clones in each group.
Figure 4
Figure 4
Long-term assessment of VCN, βAS3-globin mRNA, and HbAS3 expression in peripheral blood from secondary mice. (a) VCN analyzed in blood samples 11 weeks post-transplant. No differences in VCN were found between mice treated with the two different LV (P = 0.65, two-sample t-test). (b) Percentage of βAS3-globin mRNA per VCN from the samples shown in (a). Expression values were higher in the group of mice treated with Ank-R LV (P = 0.001, two-sample t-test). (c) The percentages of βAS3-globin mRNA per VCN, expressed in the primary and secondary mice at week 20 and 11 post-transplant, respectively, were analyzed for each LV (two-sample t-test). FB LV: primary mice, n = 9 and secondary, n = 18; for Ank-R LV-treated mice: primary, n = 8 and secondary, n = 21. The data shown in this graph at week 20 of the primary mice correspond to the average values shown in Supplementary Figure S5b; and the data shown at week 11 for the secondary mice are the same graphed in this panel in 4(b). (d) HPLC of blood samples from secondary recipients at week 11 post-transplant for hemoglobin tetramers analysis. The relative percentage of HbAS3 produced for each sample was calculated based on the sum total of areas under the curve for each of the primary hemoglobin peaks which included βmajor and βminor. Protein production was higher in the group of mice treated with Ank-R LV (P = 0.002, two-sample t-test). Bars represent average values and SD.
Figure 5
Figure 5
Long-term assessment of VCN and βAS3-globin mRNA expression in BM from secondary mice. (a) VCN analyzed in BM samples 11 weeks post-transplant. No differences in VCN were found between mice treated with the two different LV (P = 0.50, two-sample t-test). (b) Percentage of βAS3-globin mRNA per VCN from the samples shown in (a). Expression values were higher in the group of mice treated with Ank-R LV (P < 0.001, two-samples t-test). (c) The percentages of βAS3-globin mRNA expressed in the primary and secondary mice at week 20 and 11 post-transplant, respectively, were analyzed for each LV (two-sample t-test). FB-LV treated mice: primary mice, n = 9 and secondary mice, n = 18; Ank-R LV-treated mice: primary, n = 8 and secondary, n = 21. The data shown in this graph at week 20 for the primary mice correspond to the average values shown in Supplementary Figure S6, (right y-axis); and the data shown at week 11 for the secondary mice are the same as graphed in this panel in 5(b). Bars represent average and SD values.

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