Efficient Genetic Safety Switches for Future Application of iPSC-Derived Cell Transplants.

Induced pluripotent stem cell (iPSC)-derived cell products hold great promise as a potential cell source in personalized medicine. As concerns about the potential risk of graft-related severe adverse events, such as tumor formation from residual pluripotent cells, currently restrict their applicability, we established an optimized tool for therapeutic intervention that allows drug-controlled, specific and selective ablation of either iPSCs or the whole graft through genetic safety switches. To identify the best working system, different tools for genetic iPSC modification, promoters to express safety switches and different safety switches were combined. Suicide effects were slightly stronger when the suicide gene was delivered through lentiviral (LV) vectors compared to integration into the AAVS1 locus through TALEN technology. An optimized HSV-thymidine kinase and the inducible Caspase 9 both mediated drug-induced, efficient in vitro elimination of transgene-positive iPSCs. Choice of promoter allowed selective elimination of distinct populations within the graft: the hOct4 short response element restricted transgene expression to iPSCs, while the CAGs promoter ubiquitously drove expression in iPSCs and their progeny. Remarkably, both safety switches were able to prevent in vivo teratoma development and even effectively eliminated established teratomas formed by LV CAGs-transgenic iPSCs. These optimized tools to increase safety provide an important step towards clinical application of iPSC-derived transplants.

Genetic Correction of IL-10RB Deficiency Reconstitutes Anti-Inflammatory Regulation in iPSC-Derived Macrophages.

Patient material from rare diseases such as very early-onset inflammatory bowel disease (VEO-IBD) is often limited. The use of patient-derived induced pluripotent stem cells (iPSCs) for disease modeling is a promising approach to investigate disease pathomechanisms and therapeutic strategies. We successfully developed VEO-IBD patient-derived iPSC lines harboring a mutation in the IL-10 receptor ß-chain (IL-10RB) associated with defective IL-10 signaling. To characterize the disease phenotype, healthy control and VEO-IBD iPSCs were differentiated into macrophages. IL-10 stimulation induced characteristic signal transducer and activator of transcription 3 (STAT3) and suppressor of cytokine signaling 3 (SOCS3) downstream signaling and anti-inflammatory regulation of lipopolysaccharide (LPS)-mediated cytokine secretion in healthy control iPSC-derived macrophages. In contrast, IL-10 stimulation of macrophages derived from patient iPSCs did not result in STAT3 phosphorylation and subsequent SOCS3 expression, recapitulating the phenotype of cells from patients with IL-10RB deficiency. In line with this, LPS-induced cytokine secretion (e.g., IL-6 and tumor necrosis factor-α (TNF-α)) could not be downregulated by exogenous IL-10 stimulation in VEO-IBD iPSC-derived macrophages. Correction of the IL-10RB defect via lentiviral gene therapy or genome editing in the adeno-associated virus integration site 1 (AAVS1) safe harbor locus led to reconstitution of the anti-inflammatory response. Corrected cells showed IL-10RB expression, IL-10-inducible phosphorylation of STAT3, and subsequent SOCS3 expression. Furthermore, LPS-mediated TNF-α secretion could be modulated by IL-10 stimulation in gene-edited VEO-IBD iPSC-derived macrophages. Our established disease models provide the opportunity to identify and validate new curative molecular therapies and to investigate phenotypes and consequences of additional individual IL-10 signaling pathway-dependent VEO-IBD mutations.

Knockout-Induced Pluripotent Stem Cells for Disease and Therapy Modeling of IL-10-Associated Primary Immunodeficiencies.

Samples from patients with rare diseases, such as primary immunodeficiencies, are often limited, which hampers careful analysis of the pathomechanisms involved in immune cell dysregulation. To overcome this issue, induced pluripotent stem cells (iPSCs) represent an almost inexhaustible cell source and thus provide an excellent opportunity to generate disease models for rare diseases and to validate new therapeutic approaches. To obtain a better understanding of primary immunodeficiencies associated with the interleukin (IL)-10 signaling pathway, for example, very-early-onset inflammatory bowel disease (VEO-IBD), we generated genetic knockouts (KOs) of IL-10RA (IL-10 receptor α-chain) and IL-10RB (IL-10 receptor ß-chain) as well as the downstream targets of the IL-10-receptor (IL-10R) signal transducers and activators of transcription (STAT)1 and STAT3 via an sgRNA (single-guide RNA)-CRISPR-Cas9-expressing lentiviral system. IL-10 signaling-associated KO models and a VEO-IBD patient-derived iPSC clone were differentiated into macrophages for disease models. IL-10R- or STAT3-deficient disease models showed no IL-10-induced BCL3 or SOCS3 expression, whereas lipopolysaccharide (LPS) stimulation induced IL-10R independently of BCL3 and SOCS3 expression. Cytokine secretion profiles from iPSC-derived macrophage disease models showed that IL-10 was involved in many inflammatory cytokine secretions, which indicated formation of both anti- and proinflammatory macrophage phenotypes. Macrophage-secreted cytokines were separated into IL-10R- and STAT3-dependent (IL-6, TNF-α), or into IL-10R-, STAT1-, and STAT3-dependent cytokines (CCL2, CXCL10). Importantly, lentiviral correction restored IL-10-mediated regulation of LPS-induced cytokine secretion in corrected IL-10RB, STAT1, and VEO-IBD patient-derived disease models. Furthermore, treatment of IL-10RB-deficient macrophages with anti-inflammatory small molecules (SB202190, filgotinib) reduced proinflammatory cytokine secretion patterns. Taken together, the described iPSC KO models gave new insights into the pathomechanisms of immune cell dysregulation and served as model systems to test potential therapeutic approaches, including lentiviral gene therapy and targeted small-molecule treatment.