Systemic leukocyte-directed siRNA delivery revealing cyclin D1 as an anti-inflammatory target

Abstract

Cyclin D1 (CyD1) is a pivotal cell cycle-regulatory molecule and a well-studied therapeutic target for cancer. Although CyD1 is also strongly up-regulated at sites of inflammation, its exact roles in this context remain uncharacterized. To address this question, we developed a strategy for selectively silencing CyD1 in leukocytes in vivo. Targeted stabilized nanoparticles (tsNPs) were loaded with CyD1-small interfering RNA (siRNA). Antibodies to beta(7) integrin (beta(7) I) were then used to target specific leukocyte subsets involved in gut inflammation. Systemic application of beta(7) I-tsNPs silenced CyD1 in leukocytes and reversed experimentally induced colitis in mice by suppressing leukocyte proliferation and T helper cell 1 cytokine expression. This study reveals CyD1 to be a potential anti-inflammatory target, and suggests that the application of similar modes of targeting by siRNA may be feasible in other therapeutic settings.

Fig. 1

The processes involved in generating I-tsNPs. Multilamellar vesicle (MLV) [prepared as described in (9)] is extruded to form a unilamellar vesicle (ULV) with a diameter of ∼100 nm. Hyaluronan is covalently attached to DPPE in the ULV. A monoclonal antibody (mAb) to the integrin is covalently attached to hyaluronan, generating I-tsNP. siRNAs are entrapped by rehydrating lyophilized β₇ I-tsNP with water containing protamine-condensed siRNAs.

Fig. 2

β₇ I-tsNP delivers siRNAs in leukocytes in a β₇-specific manner. (A) Cy3-siRNA delivery via β₇ I-tsNP to WT, but not to β₇ knockout (KO), splenocytes as revealed by flow cytometry. (B) Confocal microscopy with differential interference contrast (DIC) morphologies showing the β₇ integrin-specific intracellular delivery of Cy3-siRNA. Images were acquired 4 hours after addition to splenocytes of naked Cy3-siRNA or Cy3-siRNA in Alexa 488-labeled β₇ I-tsNPs or IgG-sNPs. (C) Ku70-siRNA delivery with β₇ I-tsNP-induced silencing. Splenocytes were treated for 48 hours with 1000 pmol of Ku70-siRNAs or control luciferase (Luci)-siRNAs, delivered as indicated. (D) In vivo silencing of Ku70 in mononuclear cells from the gut and spleen of WT, but not KO, mice. siRNAs (2.5 mg/kg) entrapped as indicated were intravenously injected. Seventy-two hours after injection, Ku70 protein expression was determined by immunofluorescent cytometry after cell permeabilization and expressed as a percentage of Ku70 expression in mock-treated samples [(C) and (D)]. Data are expressed as the mean ± SEM of at least three independent experiments [(A), (C), and (D)]. *P < 0.05, ^†P < 0.01 versus mock-treated samples. (E) Biodistribution of ³H-cholesterylhexadecylether (³H-CHE)-labeled nanoparticles in mice with or without DSS-induced colitis. Pharmacokinetics and biodistribution were determined 12 hours after injection in a total of six mice per group in three independent experiments. Half-lives of β₇ I-tsNP in the blood of healthy and diseased mice were 4.3 and 1.8 hours, respectively. Preferential redistribution of β₇ I-tsNP to the inflamed gut is potentially advantageous for delivering siRNAs to treat intestinal inflammation. ^†P < 0.01.

Fig. 3

Silencing of CyD1 by siRNA delivery with β₇ I-tsNPs and its effects on cytokine expression. (A) Silencing of CyD1 [measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR)] and its effects on proliferation (measured by [³H]-thymidine incorporation). In in vitro treatments, splenocytes were examined after 72 hours’ incubation with 1000 pmol of siRNAs delivered as indicated in the presence or absence of CD3/CD28 stimulation. In in vivo treatments, siRNAs (2.5 mg/kg) entrapped as indicated were intravenously injected into a total of six mice per group in three independent experiments. Seventy-two hours later, mononuclear cells harvested from the gut and spleen were examined. *P < 0.05, ^†P < 0.01 versus mock-treated samples. (B) CyD1-knockdown selectively suppresses T_H1 cytokine mRNA expression in splenocytes activated via CD3/CD28. (C) CyD1-knockdown selectively suppresses T_H1 cytokine mRNA expression independently of its inhibitory effects on the cell cycle. In aphidicolin-treated TK-1 cells, in which the cell cycle was arrested, PMA/ionomycin up-regulated T_H1 cytokine mRNA expression was selectively suppressed by CyD1-knockdown. (D) Cell cycle-independent suppression of T_H1 cytokines observed with individual applications of four different CyD1-siRNAs. (C) to (D) TK-1 cells were first treated for 12 hours with aphidicolin then with siRNAs (1000 pmol) delivered as indicated for another 12 hours in the presence of PMA/ionomycin and aphidicolin. (B) to (D) *P < 0.05, ^†P < 0.01 versus mock-treated activated cells. (A) to (D) mRNA levels for CyD1 and cytokines were measured by qRT-PCR. Data are expressed as the mean ± SEM of at least three independent experiments.

Fig. 4

Cyclin-D1-siRNA delivered by β₇ I-tsNP alleviated intestinal inflammation in DSS-induced colitis. Mice were intravenously administered CyD1- or luciferase (Luci)-siRNAs (2.5 mg/kg) entrapped in either β₇ I-tsNPs or IgG-sNPs, or naked CyD1-siRNA (2.5 mg/kg) at days 0, 2, 4, and 6 (a total of six mice per group in three independent experiments). (A) Change in body weight. (B) Hematocrit (HCT) measured at day 9. (C) Representative histology at day 9 (hematoxylin and eosin staining, magnification ×100). (D) mRNA expression of CyD1 and cytokines in the gut. mRNA expression was measured by qRT-PCR with homogenized colon samples harvested at day 9. (A), (B), and (D) Data are expressed as the mean ± SEM of three independent experiments. *P < 0.05, ^†P < 0.01 versus mock-treated mice with DSS-induced colitis.