Glutamate-Cysteine Ligase Modifier Subunit Overexpression Confers Resistance Against Busulfan
Conference: ASGCT 23rd Annual Meeting
Project: Chemoprotection in Gene-Modified HSPC Transplants
- Increased expression GCL modifier subunit alone confers substantial chemoprotection against busulfan.
- Seraph Research Institute · Translational Research
- Glutamate-Cysteine Ligase Modifier Subunit Overexpression Confers Resistance Against Busulfan
- Serhat Gümrükcü 1, Phillip A. Musikanth 1, Tung Nguyen 1,2
- 1 Seraph Research Institute, Los Angeles, CA; 2 Enochian Biosciences, Los Angeles, CA
- Glutamate-Cysteine Ligase Modifier Subunit Overexpression Confers Resistance Against Busulfan
- Serhat Gümrükcü 1, Phillip A. Musikanth 1, Tung Nguyen 1,2
- 1 Seraph Research Institute, Los Angeles, CA; 2 Enochian Biosciences, Los Angeles, CA
BACKGROUND
- Lack of sufficient engraftment in gene-modified hematopoietic stem/progenitor cells (HSPCs) therapies has been the biggest challenge.
- Usage of chemo-resistance gene against cytotoxic agents select for these cells in vivo has been proposed.
- Clearance of busulfan -commonly used cytotoxic chemotherapy agent for conditioning regimens in HSPC transplants- is achieved by Glutathione (GSH) conjugation. Increased levels of GSH in the cytoplasm mediates busulfan toxicity.
- The first step and rate-limiting enzyme of GSH synthesis pathway involves glutamate-cysteine ligase (GCL)(Fig. 1).
- We evaluated whether increasing the GCL enzymatic activity through overexpressing its modifier subunit GCLM would confer protection against busulfan toxicity in vitro.
METHODS
- 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
RESULTS
- 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)
DISCUSSION
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.
CONCLUSION
One of the biggest obstacles in gene-modified autologous HSPC transplants is lack of sufficient engraftment.
- Busulfan is an agent that has higher toxicity on bone marrow stem cells and limited toxicity to other organs.
- In gene-modified autologous non-myeloablative bone marrow transplants, GCLM gene could be utilized to increase the engraftment rate of gene-modified stem cells
Corresponding author Serhat Gumrukcu
Seraph Research Institute Los Angeles, CA
drs@seraphinstitute.org
