UM171 enhances fitness and engraftment of gene-modified hematopoietic stem cells from patients with sickle cell disease

Abstract

Hematopoietic stem cell (HSC) transplantation with lentiviral vector (LVV)-transduced autologous cells has proven an effective therapeutic strategy for sickle cell disease (SCD). However, ex vivo culture or proliferative stress associated with in vivo reconstitution may amplify any underlying genetic risk of leukemia. We aimed to minimize culture-induced stress and reduce genomic damage during ex vivo culture and enhance stem cell fitness and reconstitution of SCD CD34+ cells transduced with BCL11A shmiR-encoding LVV. UM171, a pyrimidoindole derivative, can expand normal HSCs during in vitro culture and has been shown to be safe and effective using umbilical cord blood. We examined the effect of UM171 during ex vivo LVV transduction of SCD HSCs. Culture of SCD CD34+ HSCs with UM171 during transduction reduced DNA damage and reactive oxygen species, decreased apoptosis, and was associated with increased numbers of immunophenotypically defined long-term HSCs. UM171 increased the engraftment of LVV-transduced human HSCs in immunodeficient mice and barcode tracing revealed increased clonal diversity of engrafting cells. In competitive transplantation assays, analysis of bone marrow showed that cells transduced in the presence of UM171 consistently outcompeted those transduced under control conditions. In summary, exposure of SCD peripheral blood CD34+ cells to UM171 during LVV transduction enhances stem cell fitness. These findings suggest manufacturing of genetically modified HSCs in the presence of UM171 may improve efficacy, safety, and sustainability of gene therapy using ex vivo approaches. BCL11A shmiR-encoding LVV is in clinical trials to treat SCD (NCT03282656), UM171 is in clinical trials to culture umbilical cord blood (NCT02668315).

Graphical abstract

Figure 1.

UM171 enhances the in vitro expansion of CD34⁺ cells from patients with SCD. (A) Outline of in vitro manipulations and analysis. CD34⁺ cells from patients with SCD were prestimulated and transduced with LV-BCL11A in the presence or absence of UM171 and cultured 7 days for readouts. See “Material and methods” for details. (B) Total cell numbers of CD34⁺ cells transduced with LV-BCL11A in presence or absence of UM171. (C) The percentage of CD34⁺ CD133⁺ cells and (D) CD34⁺ CD133⁺ absolute cell numbers after transduction in the presence or absence of UM171. (E) The percentage of CD34⁺ CD133⁺ CD90⁺ CD45RA^– cells and (F) absolute numbers of CD34⁺ CD133⁺ CD90⁺ CD45RA^– after transduction in presence or absence of UM171. Data represent mean ± standard deviation (SD), n = 3, 3 independent experiments from cells of different patients with SCD. ∗∗P < .01; ∗∗∗P < .001.

Figure 2.

UM171 enhances the efficiency of gene transfer into CD34⁺ from patients with SCD and reduces manufacturing stress in vitro. (A) Lentiviral VCN in CD34⁺ cells transduced with LV-BCL11A in the presence or absence of UM171 was measured by quantitative PCR. (B) DNA damage was assessed by the level of γH2AX staining. (C) Assessment of the level of ROS⁺ cells, (D) cell cycle, and (E) the percentage of Annexin V⁺ cells after transduction in the presence or absence of UM171. Readouts after 7 days in culture. Data represent mean ± SD, n = 3, 3 independent experiments from cells of different patients with SCD. ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001.

Figure 3.

UM171 increases engraftment of CD34⁺ cells in xenografts. (A) Outline of assay. CD34⁺ cells from patients with SCD were prestimulated and transduced with LV-GFP in the presence or absence of UM171 and transduced cells were injected into NBSGW mice. (B) Engraftment of CD34-derived CD45⁺ cells and (C) gene-marked CD45⁺ cells in BM of NBSGW mice at week 16 after transplant. (D) Lineage distribution and (E) the percentage of gene-marked human cells from BM. Data represent mean ± SD, n = 6, each data point represents an individual mouse. ∗P < .05; ∗∗P < .01.

Figure 4.

UM171 enhances reconstitution of CD34⁺ cells expressing a BCL11A shmiR in NBSGW mice. (A) Outline of competitive assay, CD34⁺ cells from patients with SCD (n = 3) were prestimulated and transduced in the presence or absence of UM171 with a Venus- or a BFP-expressing vector. Equal numbers of Venus-marked and BFP-marked cells were mixed 1:1 and coinjected into NBSGW mice. (B) Competitive repopulation between cells transduced in presence or absence of UM171. Both vectors expressed the BCL11A shmiR. (C) Ratio of engrafted gene-marked cells after transduction in the presence or absence of UM171. Data represent mean ± SD, n = 9, each data point represents an individual mouse. ∗P < .05; ∗∗∗∗P < .0001.

Figure 5.

Effect of UM171 on clonality of barcoded engrafted BM cells. (A) Distribution of barcode frequencies detected in BM cells from transplanted NBSGW mice. Each color represents a distinct barcode. (B) Assessment of the number of unique clones from NBSGW mouse BM cells transplanted with CD34⁺ cells from different patients with SCD (noted P1-P8). Each data point represents an individual mouse transplanted with different patient’s cells. (C) The percentage of contributions of top 5 clones to engrafted BM harvested from transplanted mouse. (D) Shannon diversity index of barcodes from transplanted BM cells. Data represent mean ± SD, n = 8. ∗∗P < .01.