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Toxicity Studies on Intrathecal Injection of Low Dose of DMSO Used for Cryopreservation of Human Astrocytes in Mice

Tehila Sonnenfeld*, Einat Rauchbach*, Rotem Downey*, Daniel Blumenkrants, Arik Hasson, Graciela Kuperstein, Noam Kronfeld, Raanan Margalit, Kfir Molakandov, Vered Morad, Abraham Nyska, Shalom Guy Slutsky, Michel Revel, Michal Izrael*

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 Amyotrophic Lateral Sclerosis (ALS) was evaluated in an early-phase I/IIa 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 and to allow completion of safety and quality control testing before cell administration, 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 Institute of Cancer Research (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 NOD scid gamma mice (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: According to the study findings, intrathecal injection of a DMSO-containing formulation of cryopreserved AstroRx^® cells does not appear to have a toxic effect on mice.