RNA interference for improving the outcome of islet transplantation

Abstract

Islet transplantation has the potential to cure type 1 diabetes. Despite recent therapeutic success, it is still not common because a large number of transplanted islets get damaged by multiple challenges including instant blood mediated inflammatory reaction, hypoxia/reperfusion injury, inflammatory cytokines, and immune rejection. RNA interference (RNAi) is a novel strategy to selectively degrade target mRNA. The use of RNAi technologies to downregulate the expression of harmful genes has the potential to improve the outcome of islet transplantation. The aim of this review is to gain a thorough understanding of biological obstacles to islet transplantation and discuss how to overcome these barriers using different RNAi technologies. This eventually will help improve islet survival and function post transplantation. Chemically synthesized small interferring RNA (siRNA), vector based short hairpin RNA (shRNA), and their critical design elements (such as sequences, promoters, and backbone) are discussed. The application of combinatorial RNAi in islet transplantation is also discussed. Last but not the least, several delivery strategies for enhanced gene silencing are discussed, including chemical modification of siRNA, complex formation, bioconjugation, and viral vectors.

Figure 1

Instant blood-mediated inflammatory reaction.

Figure 2

Extrinsic and intrinsic pathways for islet cell apoptosis.

Figure 3

Autoimmune recurrence in type 1 diabetes and activation of T-cells in host immune rejection against transplanted islets. (A) After transplantation, β cell reactive CD8+, CD4+ T-cells, pro-inflammatory cytokines, and other molecules present in the host attack and destroy transplanted islet β cells. (B) Activation of T-cells in host immune response to transplanted islets. Antigen presenting cells (APCs) take and process antigens from donor and present antigens to host T cells to activate host immune response. Dendritic cells, macrophages, passenger leukocytes, and mononuclear cells are major APCs involved in the antigen presentation. Three signals are needed to fully activate T cells: signal 1, recognition of major histocompatibility complex (MHC) and the bound peptide by the T-cell receptor on the host T cells; signal 2, interaction of co-stimulation signals (such as CD40, CD80/86) with their corresponding receptors on host T cells; signal 3, soluble cytokines (IL-12, IL-10, IL-2) further stimulate the proliferation and differentiation of T cells. These interacting cell surface molecules are also RNAi targets for reduced immune rejection.

Figure 4

Combinatorial RNAi strategies for islet genetic modification. (A) siRNA pool targeting different sites of a single gene or targeting multiple genes. (B) Co-expression of multiple shRNAs, (I) multiple shRNAs under separate promoters, (II) miRNA cluster mimics, (III) long hairpin RNA. (C) Co-expression of shRNAs and cDNAs, (I) from a single miRNA backbone, (II) from separate promoters.

Figure 5

Bipartite Vectors for co-expression of VEGF cDNA and shRNA against iNOS gene. Silencing of iNOS gene expression with a bipartite vector: (a) with two separate promoters, (b) with shRNA embedded in miRNA backbone and controlled under a single promoter. (c) VEGF expression levels of bipartite vectors. Modified from [115]

Figure 6

Delivery strategies for gene silencing. Cationic liposomes and polymers are commonly used as transfection reagents for siRNA and shRNA. To avoid the use of cationic carriers, siRNA can also be conjugated to polymers such as poly(ethylene glycol) carrying targeting ligand(s). For enhanced and/or prolonged gene silencing, shRNA is often cloned into a viral (adenovirus, lentivirus, or adeno associate virus) vector. After administration, these delivery systems enter the cells mainly through endocytosis. Vector based shRNA is further translocated into cell nucleus, where shRNA is expressed to produce pri-shRNA, which is further processed into pre-shRNA by an enzyme named Drosha. Pre-shRNA is exported to the cytoplasm by exportin-5, where it is processed by Dicer (an RNAse III enzyme) to produce functional siRNA. For siRNA bioconjugation or siRNA complex, the escape and release of free siRNA from endosome into cytoplasm is an essential step. Finally, siRNAs will be incorporated into RNA-induced silencing complex (RISC) and guide the recognition and degradation of target mRNA.

Figure 7

Common modification of introduced to siRNAs. (A) Modification of non-bridging oxygen internucleotide linkage. (B) Modification of the sugar unit.

Figure 8

Transfection efficiency of siRNA into intact human islets. (A) Fluorescence is seen throughout islet surfaces with some concentrations on edges and interior surface. (B)Following transfection with FITC-labeled siRNA human islets were analyzed by flow cytometry after dispersing into single cells by trypsinization. Compared to the background fluorescence in control group, 21.5 & 28.3% of islet cells incorporated siRNA at 100 & 400 nM concentrations, respectively. Reprinted from [113].

Figure 9

Silencing of caspase-3 gene with siRNA and adenoviral shRNA. (a) Effect of caspase-3 siRNA on caspase-3/7 activity in INS-1E cells. After transfection of INS-1E cells with siRNA/Lipofectamine 2000 complexes, the cells were incubated with the cytokine cocktail for additional 16 h. Short term transient caspase 3 were achieved which lasted up to 3 days transfection. (b) Adv-H1-caspase-3-shRNA silencing effect on caspase 3/7 activity in human islets. Upon transduction, islets were incubated in the fresh medium without cytokines, then were collected at the indicated days for determining caspase 3/7 activity. The silencing effect lasted over 5 days post transduction. Modified from [105].