• We evaluated whether increasing the GCL enzymatic activity
through overexpressing its modifier subunit GCLM would
confer protection against busulfan toxicity in vitro.
• Jurkat, CEM, and TF-1a cell lines were transduced with a lentiviral vector
that expresses GCLM and green fluorescent protein (GFP) genes (Fig. 2A).
• Chemoprotection experiments were performed by incubating
untransduced or mixed populations of (20% transduced, 80% untransduced) cells with busulfan in concentrations ranging from low (2 to 10 ug/ml) to moderate and high (10 to 200 ug/ml) for 1 or 2 hours. Cells were then incubated and viable cell counts were documented in
busulfan-free culture for the 24, 48, 72 and 96-hr timepoints (Fig. 2B).
• Pre- and post-transduction, and post-exposure GSH levels were measured with fluorometric assays by FACS analysis.
• Cell survival and proliferation were measured daily by Nucleocounter NC- 200 and flow cytometry using PI and 7-AAD staining protocols. The Chemoselection
• GSH level increased in transduced Jurkat, CEM and TF-1a cells (Fig. 3A). There was no effect on cell viability and proliferation due to the overexpression of GCLM (Fig. 3B)
• GSH levels in transduced cells remained stable throughout the experiment, whereas GSH in untransduced cells was depleted after busulfan exposure (Fig. 4)
• Increased GSH activity in GLCM-transduced-CEM cells rendered at least 3.5-fold protection against busulfan at lower doses (Fig. 5)
• At higher doses (10 or 20 ug/ml) of busulfan exposure, it also exhibited protection and proliferation of the transduced cell populations (Fig. 6)
• Moderate levels of busulfan exposure achieved selection of
chemo-protected cells and 100% transduced cell population in 72- 96h (Fig. 7)
Increased expression GCL modifier subunit alone confers substantial chemoprotection against
busulfan at multiple varying doses and times of exposure.
• This indicates that GCLM transgene expression could be used for busulfan protection, hence
selection of genetically modified cells.
• To investigate the potential usage of GCLM overexpression in clinical applications, in vitro
chemo-protection experiments are to be done in primary human CD34+ HSPCs.
• Following in vitro experiments on primary human CD34+ HSPCs, syngeneic or humanized mouse models will be used for in vivo Proof-of-Concept.
One of the biggest obstacles in gene-modified autologous HSPC transplants is lack of sufficient
• Busulfan is an agent that has higher toxicity on bone marrow stem cells and limited toxicity to
• In gene-modified autologous non-myeloablative bone marrow transplants, GCLM gene could be utilized to increase the engraftment rate of gene-modified stem cells