Disease Modeling and Drug Discovery Using Induced Pluripotent Stem Cells From CINCA Syndrome Patients
Authors: Tanaka Takayuki , Ohta Akira , Yanagimachi Masakatsu , Nishikomori Ryuta , Heike Toshio , Nakahata Tatsutoshi , Yamanaka Shinya , Saito Megumu 
Published: October 2019

Abstract
Abstract 4680
Chronic infantile neurologic cutaneous and articular syndrome (CINCA syndrome; MIM #607715) is a dominantly inherited autoinflammatory disease characterized by systemic inflammation with an urticaria-like rash, neurological manifestations, and arthropathy. NLRP3 mutation is the first and so far the only identified mutation that is responsible for CINCA syndrome. NLRP3 is expressed mainly in myelomonocytic lineage cells and chondrocytes, and acts as an intracellular sensor of danger signals from various cellular insults. In normal macrophages, a first stimulus, such as lipopolysaccharide (LPS), induces the synthesis of NLRP3 and the biologically inactive proIL-1β. A second stimulus, such as ATP, enhances the assembly of a protein complex called the NLRP3-inflammasome. The inflammasome contains caspase1, which executes the proteolytic maturation and secretion of IL-1β. While normal monocytes/macrophages show no or limited IL-1β secretion in response to LPS stimulation alone, CINCA patients' cells exhibit robust IL-1β secretion, because the mutant NLRP3-inflammasome is auto-activated without the need for any second stimulus.
While approximately half of CINCA patients carry heterozygous gain-of-function mutations of the NLRP3 gene, 30 to 40% of all patients have mutations in NLRP3 in only a small number of somatic cells. Since the population of mutant cells is relatively small (4.2–35.8% in blood cells), it remains controversial whether the small fraction of NLRP3-mutated cells actually causes the strong autoinflammation observed in CINCA patients, or whether the NLRP3 mutations found in mosaic patients are just a bystander, with all cells carrying an unknown mutation of another gene that causes the disease. The pathogenesis of CINCA syndrome patients who carry NLRP3mutations as somatic mosaicism has not been precisely described because of the difficulty in separating live cells based on the presence or absence of the mutation.
It is believed that most of the manifestations of CINCA syndrome are caused by the excessive secretion of the proinflammatory cytokine, IL-1β, and this concept is supported by the efficacy of an IL-1 receptor antagonist (IL-1Ra) for decreasing most of the symptoms. However, an NLRP3-targeted therapeutic approach would be attractive because 1) the progressive arthropathy despite anti-IL-1 therapy indicates that the presence of additional proteins processed by the inflammasome is also involved in the pathogenesis of CINCA syndrome 2) specific inhibition of the NLRP3-inflammasome can avoid unfavorable suppression of other IL-1β processing pathways in response to various triggers, and 3) these drugs may be also effective for various other NLRP3-related chronic inflammatory conditions, such as Alzheimer's disease, diabetes, severe gout and atherosclerosis.
Here, we report the generation of NLRP3-mutant and non-mutant induced pluripotent stem cell (iPSC) lines from two CINCA syndrome patients with somatic mosaicism, and describe their differentiation into macrophages (iPS-MPs). We found that mutant cells are predominantly responsible for the pathogenesis in these mosaic patients because only mutant iPS-MPs showed the disease relevant phenotype of abnormal IL-1β secretion.
Next, after confirming that the existing anti-inflammatory compounds inhibited the abnormal IL-1β secretion from iPS-MPs, we started drug screening for CINCA syndrome and other NLRP3-related inflammatory conditions using a recently-established feeder-free differentiation protocol. Among the 3,939 chemically-potent low molecule compounds, 127 candidates inhibited IL-1β secretion by more than 30%. We then excluded compounds that also inhibited IL-6 secretion and selected 23 candidate compounds. We are now validating the potency of these compounds.
Our results illustrate that patient-derived iPSCs are useful for dissecting somatic mosaicism, and that NLRP3-mutant iPSCs can provide a valuable platform for drug discovery for multiple NLRP3-related disorders.

Disclosures:
Yamanaka: iPierian: Membership on an entity's Board of Directors or advisory committees.