Simon N Chu, United States has been granted the TTS-IPITA Congress Scientific Award
Improving the safety of stem-cell-derived beta cell transplantation with an inducible safety switch
Simon N Chu1,4,5, N Shabrina Amirruddin2,3,4,5, Devesh K Sharma1,5, Isabel C Sierra2,3,4,5, Peter G Stock1, Julie B Sneddon2,3,4,5, M Kyle Cromer1,5,6.
1Department of Surgery, University of California, San Francisco, San Francisco, CA, United States; 2Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, United States; 3Department of Anatomy, University of California, San Francisco, San Francisco, CA, United States; 4Diabetes Center, University of California, San Francisco, San Francisco, CA, United States; 5Eli & Edythe Broad Center for Regeneration Medicine, University of California, San Francisco, San Francisco, CA, United States; 6Department of Bioengineering & Therapeutic Sciences, University of California, San Francisco, San Francsico, CA, United States
Introduction: In patients with type 1 diabetes mellitus, glucose homeostasis can be re-established with allogeneic islet transplantation. This approach has the advantage of overcoming the surgical morbidity of whole pancreas transplant. However, widespread applicability of both therapies is limited by a paucity of deceased donors. Pluripotent stem cell-derived islets offer a potentially renewable source of functional beta cells, but a major limitation of stem cell-derived therapies is the risk of teratoma formation following transplantation. Therefore, for stem cell-derived islet transplantation to reach its full therapeutic potential, safety concerns over iatrogenic tumorigenesis and teratoma formation must be overcome. Here, we describe the results of a CRISPR/AAV-mediated genome engineering strategy to integrate an inducible safety switch into the HUES8 embryonic stem cell line. This approach may impart a drug-inducible safeguard into stem cell-derived beta cells, allowing for eradication of the complete allograft in the case of an adverse event.
Methods: We identified a Cas9 guide RNA specific to the 3’ UTR of the ACTB housekeeping gene, which is ubiquitously expressed in all cells. We then designed an iCaspase9-mPlum expression cassette, whose expression is induced by the small molecule A/C heterodimerizer, AP21967. This cassette was then integrated by an AAV6 vector into luciferase-expressing HUES8 cells, linking expression of our integration cassette to ACTB expression. Edited HUES8 cells were sorted by fluorescence activated cell sorting and expanded in vitro. Small molecule was then added and killing of edited cells was assessed by flow cytometry.
Results: HUES8 cells were successfully edited as seen by mPlum positivity on flow cytometry, with both monoallelic and biallelic edited populations obtained. Addition of AP21967 led to killing of both populations, demonstrating the ability to induce apoptosis in edited cells expressing our transgene cassette via the control of a small molecule.
Conclusions: We demonstrate successful editing of HUES8 embryonic stem cells and integration of an inducible safety switch into the ACTB locus, allowing inducible killing of pluripotent cells. Further testing to validate that genome engineering has not compromised efficiency of downstream stem cell-derived beta cell differentiation or function is underway. These encouraging findings support further testing to validate the ability of this ACTB-driven switch to reduce teratoma risk of edited stem cell-derived beta cells using in vivo mouse transplantation experiments.
[1] Pagliuca FW, Millman JR, Gürtler M, et al. Generation of functional human pancreatic β cells in vitro. Cell. 2014;159(2):428-439. doi:10.1016/j.cell.2014.09.040
[2] Fujikawa T, Oh SH, Pi L, Hatch HM, Shupe T, Petersen BE. Teratoma Formation Leads to Failure of Treatment for Type I Diabetes Using Embryonic Stem Cell-Derived Insulin-Producing Cells. Am J Pathol. 2005;166(6):1781-1791.
[3] Lee AS, Tang C, Rao MS, Weissman IL, Wu JC. Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies. Nat Med. 2013;19(8):998-1004. doi:10.1038/nm.3267
[4] Tang C, Weissman IL, Drukker M. The Safety of Embryonic Stem Cell Therapy Relies on Teratoma Removal. Oncotarget. 2012;3(1):7-8.
[5] Han L, He H, Yang Y, et al. Distinctive Clinical and Pathologic Features of Immature Teratomas Arising from Induced Pluripotent Stem Cell-Derived Beta Cell Injection in a Diabetes Patient. Stem Cells Dev. 2022;31(5-6):97-101. doi:10.1089/scd.2021.0255
[6] Martin RM, Fowler JL, Cromer MK, et al. Improving the safety of human pluripotent stem cell therapies using genome-edited orthogonal safeguards. Nat Commun. 2020;11(1):2713. doi:10.1038/s41467-020-16455-7
