Islet transplantation is a challenging treatment for type-1 diabetic patients. This is due, in part, to the deleterious effects of pro-inflammatory cytokines generated by early host innate immune responses that reduce islet engraftment and function. These cytokines activate intra-islet NF-ΚB, leading to the production of iNOS, Fas and chemokines detrimental to Β cell survival and function. We hypothesized that inhibition of IKKΒ, a kinase subunit crucial for activation of NF-ΚB, would protect against cytokine-induced dysfunction and enhance transplanted islet survival.
To test our hypothesis, siRNA-based spherical nucleic acid gold nanoparticle conjugates were prepared using a novel siRNA sequence targeting IKKΒ (SNA-NC-IKKΒ). Freshly isolated mouse islets were treated in culture with 10 nM SNA-NC-IKKΒ for 24 hours.
SNA-NC-IKKΒ penetrated to the core of the islets and decreased IKKΒ expression to 49.1% compared to untreated islets as assessed by RT-qPCR. siRNA knockdown of IKKΒ reduced activation of NF-ΚB when islets were exposed in vitro to cytokines (50U/mL IL-1Β, 1000 U/mL TNF-Α, 750 U/mL IFN-Γ). Further, SNA-NC-IKKΒ treatment prevented cytokine-induced expression of iNOS, Fas and chemokines compared to controls. In addition, SNA-NC-IKKΒ protected Β cells from cytokine-induced cell death. The viability and percentage of Β cells, assayed by flow cytometry, was significantly higher in SNA-NC-IKKΒ treated islets (81.2% ± 2.03%; 42.7% ± 2.63% respectively) versus controls (60.7% ± 2.57%, p<0.05; 35.3% ± 0.90%, p<0.05). Syngeneic intra-portal transplants of a marginal mass of 50 islets to streptozotocin-induced diabetic mice resulted in reversion to normoglycemia in 50% of SNA-NC-IKKΒ treated islet recipients on day 5.67 ± 2.5 (n=12) versus 0% of control recipients (n=12; p<0.05). Histologic analyses showed decreased CD11b+ cell infiltration in SNA-NC-IKKΒ islet grafts compared to controls.
The combined use of nanotechnology and gene therapy in a preconditioning strategy to treat freshly isolated islets in culture targeting IKKΒ pre-transplant protected against the detrimental effects of graft site pro-inflammatory mediators and significantly enhanced islet graft function and survival.
To cite this abstract in AMA style:Rink J, McMahon K, Zhang X, Chen X, Mirkin C, Thaxton C, Kaufman D. Knockdown of Intra-Islet IKKβ by Spherical Nucleic Acid Nanoparticle Conjugates Prevents Host Cytokine-Induced Injury and Enhances Islet Graft Survival [abstract]. Am J Transplant. 2013; 13 (suppl 5). http://atcmeetingabstracts.com/abstract/knockdown-of-intra-islet-ikk-by-spherical-nucleic-acid-nanoparticle-conjugates-prevents-host-cytokine-induced-injury-and-enhances-islet-graft-survival/. Accessed February 21, 2018.
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