Transient Bcl-XL Overexpression in Donor Stem Cells Increases Efficacy of Hematopoietic Stem Cell Transplantation without Increasing the Risk of Leukemogenesis

Abstract

Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for many hematological and immunological diseases but is hampered by the risk of graft failure or delayed engraftment. Clinical experience has shown that these problems can be overcome by transplantation of higher numbers of donor stem cells. This can be achieved by more efficient collection strategies (i.e. mobilization regimens) or by ex vivoexpansion. Here, we aim to transiently inhibit apoptosis in donor hematopoietic stem and progenitor cells (HSPCs) prior to transplantation in order to increase their numbers and their fitness. In previous studies we have identified two Bcl-2 proteins from the pro-apoptotic BH3-only subgroup, Bim and Bmf, to be central players in apoptosis induction of HSPCs during transplantation. Both proteins are efficiently repressed by the cytokines Flt3L and SCF and upregulated under cytokine deprivation in vitro. Lack of either protein or overexpression of their anti-apoptotic antagonists Bcl-2 or Bcl-xL strongly increased HSPC competitiveness during transplantation, both in murine transplantation and human xenotransplantation models. Moreover, less donor HSPCs were required for successful engraftment when Bim-mediated apoptosis was inhibited. In sum, our data indicated that modulation of Bim or Bmf levels inhibits apoptosis in murine and human HSPCs and that the resulting extended life span is beneficial during HSCT (Labi et al, 2013). Inhibition of the intrinsic apoptosis pathway could serve as a novel therapeutic approach to increase resistance of human HSPCs to factor deprivation and other types of stress caused during HSCT. However, permanent apoptosis inhibition in HSPCs can trigger their malignant transformation over time, especially when occurring together with activation of oncogenes promoting cell proliferation. Thus, such apoptosis inhibition needs to be transient when used therapeutically. Here we analyze whether transient apoptosis resistance lasting for only a limited time span is sufficient to increase competitiveness of HSPCs during HSCT. For overexpression of Bcl-xL we used adenoviral vectors known to act transiently. In proliferating murine HSPCs, adenoviral Bcl-xL persisted for 5-7 days, and for this period cells were protected from different stress stimuli engaging the intrinsic apoptosis pathway. Most importantly, adenoviral Bcl-xL overexpression increased the reconstitution potential of murine HSPCs in competitive transplantation experiments. Persistence of adenoviruses was excluded. As expected, transient Bcl-xL overexpression did not accelerate lymphomagenesis, neither on a wildtype nor on a premalignant murine background. However, adenoviral infection was associated with a relevant amount of toxicity to murine HSPCs. We thus performed transfection of full length Bcl-xL protein coupled to a protein transduction domain. Fluorescence microscopy indicated its mitochondrial localization, and functional tests revealed protection from apoptosis. However, when compared to adenoviral overexpression, Bcl-xL protein transduction proved to be less efficient due to the short protein half-life. In vivoexperiments are ongoing and will show whether such short-term apoptosis inhibition is sufficient to increase efficacy of transplantation. In sum, our studies will evaluate the benefit of therapeutic apoptosis inhibition in donor HSPCs during HSCT and contribute to on-going efforts aiming to improve transplantation medicine. Disclosures No relevant conflicts of interest to declare.

Non-viral DNA delivery and TALEN editing correct the sickle cell mutation in hematopoietic stem cells

“…Substantial numbers of HSCs are known to be lost during transplantation due to apoptotic cell death controlled by proteins from the Bcl-2 family and a the lack of signals derived from the stem cell niche 71 . The transient overexpression of Via-Enh01 in our protocol resulted in increased viability of edited progenitors and potentially preserved the engraftment capability of edited HSCs as previously reported 31 – 33 .…”

“…2d , 71.6% ± 0.7% and 39.6% ± 4.4% average frequencies of viability ± SD, respectively) under GMP-compatible conditions compared to standard R&D conditions. We thus further optimized our gene editing process, adding an mRNA named Via-Enh01, which encodes an antiapoptotic protein 31 – 33 , to the TALEN and HDR-Enh01 mRNAs. The addition of Via-Enh01 led to a significant increase in HSPC viability compared to HDR-Enh01 alone or the standard protocol under GMP-compatible conditions (Supplementary Fig.…”

TALEN-mediated intron editing of HSPCs enables transgene expression restricted to the myeloid lineage

“…CD34+ HSPCs were transfected 48 hours after thawing and expansion by combining 5 μg of each AAVS1 TALEN mRNA or 10 μg of each CD11b TALEN mRNA with 1e6 HSPC in 100 μL BTXpress Solution (BTX Technologies, Hawthorne, NY, USA) and electroporating using the PulseAgile (Cellectis, Paris, France). One μg of Via-Enh01 mRNA and 4 μg of HDR-Enh01 mRNA were also included in the electroporation mix because Via-Enh01 39 protein inhibits cell apoptosis and because HDR-Enh01 40 increases homologous recombination by inhibiting the non-homologous-end-joining pathway. Following electroporation, cells were immediately mixed with HSPC culture media.…”

TALEN-mediated intron editing of HSPCs enables transgene expression restricted to the myeloid lineage

“…CD34+ HSPCs were transfected 48 hours after thawing and expansion by combining 5 μg of each AAVS1 TALEN mRNA or 10 μg of each CD11b TALEN mRNA with 1e6 HSPC in 100 μL BTXpress Solution (BTX Technologies, Hawthorne, NY, USA) and electroporating using the PulseAgile (Cellectis, Paris, France). One μg of Via-Enh01 mRNA and 4 μg of HDR-Enh01 mRNA were also included in the electroporation mix because Via-Enh01 39 protein inhibits cell apoptosis and because HDR-Enh01 40 increases homologous recombination by inhibiting the non-homologous-end-joining pathway. Following electroporation, cells were immediately mixed with HSPC culture media.…”