Toxicity studies on intrathecal injection of low dose of DMSO used for cryopreservation of human astrocytes in mice

Abstract

Background AstroRx® is an allogeneic cell therapy, composed of healthy and functional human astrocytes derived from pluripotent embryonic stem cells. An intrathecal injection of a fresh formulation of AstroRx® cells for the treatment of ALS was evaluated in an early-phase clinical trial. The results of this study indicated that the treatment is safe and showed a signal of a clinical benefit in attenuating ALS progression. Due to the logistical challenges associated with the manufacturing and distribution of a fresh cell product, a cryopreserved formulation of AstroRx® was developed. The cryopreseved AstroRx® product includes 3.5% DMSO as a cryoprotectant. Upon thawing at the clinical site, the cryopreserved product is diluted before its use to achieve a concentration of 0.23% DMSO. Objective To evaluate the toxicity of DMSO-containing cryopreserved AstroRx® as compared to the fresh AstroRx® following their intrathecal injection into mice. Methods In vitro compatibility assessment between cryopreserved and fresh AstroRx® formulations, including cell viability, cell number, cell identity, impurities, safety and potency, was performed. In addition, a neurotoxicity assessment of intrathecal injection of DMSO alone was tested in immunocompetent ICR mice using two concentrations of DMSO, 0.25% and 0.5%. The neurotoxicity of DMSO-containing cryopreserved AstroRx® product was evaluated in immunodeficient NSG mice. Results In-vitro comparability results demonstrated similarity between fresh AstroRx® (n = 13) and cryopreserved AsrtroRx® (n = 11) cell batches in all tested parameters. Intrathecal injection of DMSO at a concentration of 0.25% or 0.5% showed no difference, as compared to the control group, in food consumption, body weight, clinical symptoms, as well as neurological locomotor and beam tests, for 7 days post injection. Similarly, a single intrathecal injection of AstroRx® cryopreserved with DMSO following thawing or fresh AstroRx® to NSG mice was not associated with neurological signs or major systemic adverse effects during the 4- week study period. The presence of both fresh and cryopreserved AstroRx® cells at 4 weeks post injection was confirmed by Alu in-situ hybridization. Conclusion