Type 1 diabetes (T1D) is caused by the autoimmune destruction of pancreatic beta cells. Pluripotent stem cells can now be differentiated into beta cells, thus raising the prospect of a cell replacement therapy for T1D. However, autoimmunity would rapidly destroy newly transplanted beta cells. Using a genome-scale CRISPR screen in a mouse model for T1D, we show that deleting RNLS, a genome-wide association study candidate gene for T1D, made beta cells resistant to autoimmune killing. Structure-based modelling identified the U.S. Food and Drug Administration–approved drug pargyline as a potential RNLS inhibitor. Oral pargyline treatment protected transplanted beta cells in diabetic mice, thus leading to disease reversal. Furthermore, pargyline prevented or delayed diabetes onset in several mouse models for T1D. Our results identify RNLS as a modifier of beta cell vulnerability and as a potential therapeutic target to avert beta cell loss in T1D.
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This research was supported in part by funds from the Pittsburgh Foundation/Walton Fund to P.Y., funds from the Myra Reinhard Family Foundation and grants from the Harvard Stem Cell Institute (no. DP-0167-17-00), Juvenile Diabetes Research Foundation (no. 2-SRA-2018-499-S-B) and National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (no. 1R01DK120445) to P.Y. and S.K., by postdoctoral fellowships from the NIDDK (no. T32DK007260) to E.P.C. and W.Z., from the Mary K. Iacocca Foundation to E.P.C., Y.I. and W.Z., from the American Diabetes Association (no. 1-19-PMF-024) to N.C.L. and from the Japanese Society for the Promotion of Science to Y.I. We acknowledge support from core facilities funded by the NIDDK Diabetes Research Center award no. P30DK036836 to the Joslin Diabetes Center.
P.Y. and S.K. have filed patent applications related to the work described in this manuscript. D.A.M. is a scientific founder and a board observer of Semma Therapeutics. The authors declare that they have no other competing interests.
Peer review information Primary Handling Editors: George Caputa; Elena Bellafante.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Autoimmune killing of NIT-1 cells in NOD mice can be visualized by bioluminescence imaging.
a,b, Bioluminescence imaging of 107 NIT-1 cells transplanted subcutaneously into NOD.scid mice. Transplanted cells were engineered to carry a CMV-luciferase2 (Luc2) reporter. Some recipient mice were also injected intravenously with 107 splenocytes isolated from spontaneously diabetic (DM) NOD mice to cause beta cell killing. Images were taken at day 1 (a) and 15 (b) post-injection.
Extended Data Fig. 2 Generation of Rnls-mutant beta cells by CRISPR-Cas9 targeting.
a, T7 endonuclease I assay. Genomic DNA from NIT-1 wild-type (WT) and Rnlsmut cells was tested for CRISPR-Cas9 gene editing events. Cleavage at heteroduplex mismatch sites by T7 endonuclease I digestion was analyzed by agarose gel electrophoresis. DNA from Rnlsmut cells segregated into multiple digested fragments, indicating efficient mutation of the targeted region in the Rnls gene. b, Genomic DNA from Rnlsmut cells was sequenced to identify individual mutations. The Rnls gRNA targeting site is labelled in red. The frequency of the wild-type allele and of the most abundant mutations and their predicted consequence (frameshift / in-frame deletion) are shown. These frequencies indicate that 75% of the cells are predicted to carry at least one deleterious mutant allele. c, Islets (≈1700) were purified from 8-week old CD1 mice, dispersed and transduced with lentivirus encoding a non-targeting (NT) or Rnls-targeting gRNA together with the Cas9 endonuclease driven by the rat insulin promoter. 72 h later, islets were stimulated sequentially with 2.8 mM glucose, 16.8 mM glucose and finally 30 mM KCl to induce insulin secretion. Islet genomic DNA was quantified for normalization of ELISA insulin measurements to DNA content. n=5 technical replicates per condition and genotype. Data show mean ± SEM. Note that islet dispersion necessary for lentiviral transduction decreased the overall responsiveness of purified islets compared to intact islets. Insulin secretion by Rnls mutant islet cells was not significantly different from that of control (NT) islets. d, Growth curves for NIT-1 WT, control (NT gRNA) and Rnlsmut cells seeded in 96-well plates at 50,000 cells/well over one week. Culture media were refreshed in every 2 days. Cell growth was measured on days 0, 3 and 7 using the CellTiter-Glo luminescence Cell Viability Assay (Promega). Growth rates were not significantly different as calculated by one-way ANOVA with Dunnett’s multiple comparisons test. n=3 technical replicates per genotype. Data show mean ± SEM.
Extended Data Fig. 3 Rnls mutation prevents autoimmune killing but not allo-rejection of NIT-1 beta cells.
Control and Rnlsmut NIT-1 cells (107) carrying a luciferase reporter were implanted on opposing flanks of NOD.scid (a) or C57BL/6 mice (b,c). a, Graft bioluminescence was measured on days 0 and 57 after transplantation of NIT-1 cells together with diabetogenic NOD splenocytes (as in Fig. 2). b,c, Graft bioluminescence was measured on days 0, 4 and 7 after transplantation. Representative bioluminescence images (b) and relative luminescence of grafts over time (c) are shown (n=3). Data represent mean ± SEM. Both control and mutant grafts were destroyed by allo-rejection within a week.
Extended Data Fig. 4 Rnls expression modulates the sensitivity to ER stress-induced cell death but not to ER stress-unrelated apoptosis.
a, Viability of Rnlsmut and control NIT-1 cells at 6 h and 24 h after treatment with 40 mM streptozotocin (STZ) (n=3 technical replicates). b, NIT-1 cell viability 48 h after mitomycin C (MMC) at the indicated concentration (n=3 technical replicates). *** P < 0.0001, calculated by two-way ANOVA with Sidak’s multiple comparisons test. c-f, Rnls knockout NIT-1 cell lines were generated by deleting either exons 2-4 or exon 5. Deletion efficiency was confirmed by qPCR of genomic DNA. Rnls ΔEx2/4 cells showed ~60% deletion of exons 2-4 genomic DNA qPCR (c) while Rnls ΔEx5 cells showed ~87% deletion of exon 5 (d). Cell viability of Rnls deficient cells was measured 72 h after thapsigargin (TG, e) and tunicamycin (TC, f) treatment. *** P < 0.0001, calculated by unpaired t-test (c,d) and two-way ANOVA with Sidak’s multiple comparisons test (e,f). g,h, Overexpression of Rnls in WT NIT-1 cells increased sensitivity to low dose-TG-induced killing (g). n=4 technical replicates per group. *** P < 0.0001, calculated by two-way ANOVA with Sidak’s multiple comparisons test. CRISPR-immune Rnls (CiRnls) expressed in Rnlsmut cells restored sensitivity to TG-induced killing (h). n=4 technical replicates per group. ***P < 0.001, #P = 0.0138, ###P = 0.0002, 0.0005 for 0.05 and 0.25 TG(μM) respectively, calculated by two-way ANOVA with Sidak’s multiple comparisons test. *Comparison of control vs. Rnlsmut cells; #comparison of Rnlsmut vs. Rnlsmut + CiRnls cells. All data represent mean ± SEM.
Extended Data Fig. 5 Rnls overexpression increases sensitivity to autoimmune killing in vivo.
a-c, Control (WT) and Rnls overexpressing (RnlsOE) NIT-1 cells carrying a luciferase reporter were implanted on opposing flanks of NOD.scid mice. Some graft recipients were also injected intravenously with splenocytes from diabetic NOD mice (DM NOD splenocytes). Graft bioluminescence was imaged on days 0, 2, 3 and 7 (a). The relative luminescence of RnlsOE and control grafts over time, normalized to day 0, is shown in (b). Data for all mice analyzed on day 3 is shown in (c). RnlsOE graft were more sensitive to autoimmune killing as evidenced by more rapid loss of luminescence. By day 7, both control and RnlsOE grafts were killed to ~90% (data not shown), resulting in a similar relative luminescence level. n=6 mice (each with two grafts). Data represent mean ± SEM, **P < 0.0022, calculated by two-sided Mann-Whitney test. d,e, Rnlsmut NIT-1 cells and Rnlsmut cells expressing the CRISPR-immune Rnls transgene (CiRnls), all carrying a luciferase reporter, were implanted on opposing flanks of NOD.scid mice. Graft recipients were also injected intravenously with splenocytes from diabetic (DM) NOD mice. Graft bioluminescence was imaged on days 0, 2, 3 and 5 post-injection (d). Relative luminescence of paired grafts over time normalized to day 0 is shown in (e). n=5 mice. Data represent mean ± SEM.
Extended Data Fig. 6 Rnls deficiency diminishes the UPR following ER stress and protects against oxidative stress.
a, Quantification of Western blot data shown in Fig. 4e. Images were obtained and quantified using a C-DiGit scanner and the Image Studio software (LI-COR Biosciences). n=3 per group. Data show mean ± SEM, *# P < 0.05, **## P < 0.01, ***### P < 0.001, calculated by one-way ANOVA with Dunnett’s multiple comparisons test. *Comparison to control cells without TG treatment; #comparison to control cells with 5-hour TG treatment. b, Control (Ctrl) and Rnlsmut NIT-1 cells were cultured overnight with or without hydrogen peroxide (H2O2) at the indicated concentrations. Cell viability was assessed using the CellTiter-Glo luminescence Cell Viability Assay. Data show mean ± SEM of triplicate cultures and are representative of three independent experiments. **** P<0.0001, calculated by two-way ANOVA with Sidak’s multiple comparison test.
Extended Data Fig. 7 Pargyline treatment preserves insulin expression in NOD mice with long-duration diabetes.
Pancreases were isolated from control and pargyline-treated diabetic NOD mice described in Fig. 6 that were euthanised at day 20 post beta cell-transplantation. Pancreatic sections were stained with anti-insulin (DAKO, #A0564), anti-CD3 (Bio-rad, #MCA500), and DNA dye Hoechst 33342 (Invitrogen, #H3570). Goat anti-guinea pig Alexa Flour 488 and donkey anti-rat Alexa Flour 594 secondary antibodies (Thermo Fisher Scientific, #A11073 and #A21209) were used to detect insulin and CD3 antibodies, respectively. a, Representative images of individual islets, taken with a Zeiss LSM710NLO confocal microscope. b, Representative pancreas section from a pargyline (PG)-treated animal scanned using a Thermo Fisher Scientific EVOS FL Auto imaging system. Five islets were identified on the section: islets #1-4 showed many insulin-expressing cells, islet #5 had no remaining insulin-expressing cells. No significant insulin staining was detectable in the pancreas of untreated mice (not shown). c, Plasma insulin levels at day 20 post-transplantation in diabetic mice with a NIT-1 beta cell graft that were treated or not with PG. Data show mean ± SEM of n=5 mice per group and are representative of two independent experiments, * P = 0.0367, calculated by two-sided unpaired t-test.
Extended Data Fig. 8 Pargyline treatment does not prevent beta cell destruction after allo-transplantation and has no glucose-lowering effect on its own.
a, b, Wild-type NIT-1 cells (107) carrying a luciferase reporter were implanted into C57BL/6 mice that were treated or not with oral pargyline via addition to the drinking water. Graft bioluminescence was measured on days 1, 2, 3 and 4 after transplantation. Representative bioluminescence images (a) and relative luminescence of grafts over time (b) are shown. Data show mean ± SEM for n=3 mice per group. c, Pargyline did not decrease hyperglycemia in C57BL/6 mice rendered diabetic by streptozotocin (STZ) injection (150mg/kg). Data show mean ± SEM for n=5 mice (control) and n=9 (pargyline).
Extended Data Fig. 9 Pargyline prevents or delays diabetes in multiple mouse models for T1D.
a, Diabetes frequency after cyclophosphamide injection of NOD mice fed with control water (Ctrl, n=40) or water containing pargyline (PG, n=39). b, Day of disease onset in mice that developed diabetes after cyclophosphamide injection (Ctrl n=19, PG n=11). c, Diabetes frequency in NOD mice injected with blocking anti-PD-1 antibody with (n=6) or without (n=5) oral PG treatment (as in a). d, Diabetes frequency in NOD.scid mice transplanted with splenocytes (107 cells) from diabetic NOD mice and treated with or without PG (n=10 per group). e,f, Diabetes frequency (n=10 per group) and day of disease onset (ctrl n= 9, PG n=8) in C57BL/6 mice treated with multiple low doses of streptozotocin. Kaplan-Meier survival curves were compared by Log-rank test (a,c,d and e). Time of disease onset is shown as mean ± SEM and was compared by Mann-Whitney test (b,f). Exact P values are shown. g, Insulin and T cell marker staining in pancreas sections from NOD mice two weeks after anti-PD-1 injection, with or without PG treatment.
Extended Data Fig. 10 Design, genotyping and phenotyping of RNLS deletion in human SC-beta cells.
a, RNLS dual-gRNA design for the generation of RNLS knockout (KO) human induced pluripotent stem cells (SC). b, Genotyping of SC clones. CRISPR targeted clones were genotyped by PCR that was repeated for confirmation for all mutant clones, and individual mutations were verified by sequencing. Clone 1 was used as RNLS KO in this study and carried a 112bp deletion on both alleles. c, Glucose stimulated insulin secretion by SC-beta cells differentiated from WT or RNLS KO isogenic SC clones. Data in c show mean insulin secretion from four independent SC-beta cell batches, each measured in triplicate, following stimulation with 2.8mM glucose, 20mM glucose, or 30mM potassium chloride (KCl). Data show mean ± SEM for n=4 technical replicates per condition and genotype.
Source Data Fig. 4
Uncropped western blots for Fig. 4.
Source Data Fig. 5
Uncropped western blots for Fig. 5.
Source Data Extended Data Fig. 10
Uncropped agarose gel for Extended Data Fig. 10.
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Cai, E.P., Ishikawa, Y., Zhang, W. et al. Genome-scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes. Nat Metab 2, 934–945 (2020). https://doi.org/10.1038/s42255-020-0254-1
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