Delivery systems for the direct application of siRNAs to induce RNA interference (RNAi) in vivo

Abstract

RNA interference (RNAi) is a powerful method for specific gene silencing which may also lead to promising novel therapeutic strategies. It is mediated through small interfering RNAs (siRNAs) which sequence-specifically trigger the cleavage and subsequent degradation of their target mRNA. One critical factor is the ability to deliver intact siRNAs into target cells/organs in vivo. This review highlights the mechanism of RNAi and the guidelines for the design of optimal siRNAs. It gives an overview of studies based on the systemic or local application of naked siRNAs or the use of various nonviral siRNA delivery systems. One promising avenue is the the complexation of siRNAs with the polyethylenimine (PEI), which efficiently stabilizes siRNAs and, upon systemic administration, leads to the delivery of the intact siRNAs into different organs. The antitumorigenic effects of PEI/siRNA-mediated in vivo gene-targeting of tumor-relevant proteins like in mouse tumor xenograft models are described.

Figure 1

Mechanism of RNA interference (RNAi) in mammalian systems. Long double-stranded RNA molecules (dsRNA), which are expressed from DNA vectors (left red arrow) or directly enter the cell (center red arrow), are processed by the Dicer complex resulting in the formation of small inhibitory RNAs (siRNAs). Alternatively, to induce RNAi these small 21–23 bp duplexes are directly delivered into the cell (right red arrow). The siRNAs are incorporated into a nuclease-containing multiprotein complex called RISC, which becomes activated upon the ATP-dependent unwinding of the siRNA duplex by an RNA helicase. The now single-stranded siRNA guides the RISC complex to its complementary target mRNA which is then cleaved by the endonucleolytical activity of RISC. While the RISC complex is recovered for further cycles, the cleaved mRNA molecule is rapidly degraded due to its unprotected RNA ends.

Figure 2

Polyethylenimine (PEI)-mediated siRNA transfer. Upper panel: PEIs are synthetic linear (a) or branched (b) polymers with an amino group in every third position. Dependent on the pH, some of these amino nitrogens are protonated giving PEI a high cationic charge density. Lower panel: proposed mechanism of PEI-mediated siRNA transfer. Due to electrostatic interactions, PEI is able to complex negatively charged siRNAs leading to a compaction and the formation of small colloidal particles which are endocytosed. The “proton sponge effect” exhibited by PEI complexes leads to osmotic swelling and ultimately to the disruption of the endosomes. siRNAs are protected from degradation due to their tight condensation in the complex and the buffering capacity of PEI. Upon their release from the PEI-based complex, intact siRNAs are incorporated into the RISC complex and induce RNAi (see Figure 1).

Figure 3

Protection and in vivo delivery of siRNAs upon PEI complexation. In [70] (a) in vitro protection of siRNAs against nucleolytic degradation. [³²P] end-labeled siRNAs, complexed (upper panel) or not complexed (lower panel) with PEI, were subjected to treatment with 1 % fetal calf serum at 37^°C. At the time points indicated, the samples were analysed by agarose gel electrophoresis, blotting, and autoradiography. The bands represent full-length siRNA molecules indicating that PEI complexation leads to the efficient protection of siRNAs while noncomplexed siRNAs are rapidly degraded. (b,c) In vivo delivery of intact siRNAs upon PEI complexation. [³²P]-labeled siRNAs, complexed (+) or not complexed (−) with PEI, were injected IP into mice bearing subcutaneous SKOV-3 ovarian carcinoma cell tumor xenografts, and after 30 min (b) or 4 h (b) total RNA from various organ and tissue homogenates was prepared and subjected to agarose gel electrophoresis prior to blotting and autoradiography. The bands represent intact [³²P]-labeled siRNA molecules which for several hours are mainly found in tumor and muscle as well as in liver and, time-dependently, in kidney. Only little siRNA amounts are detected in the lung and traces in the brain.

Figure 3

Protection and in vivo delivery of siRNAs upon PEI complexation. In [70] (a) in vitro protection of siRNAs against nucleolytic degradation. [³²P] end-labeled siRNAs, complexed (upper panel) or not complexed (lower panel) with PEI, were subjected to treatment with 1 % fetal calf serum at 37^°C. At the time points indicated, the samples were analysed by agarose gel electrophoresis, blotting, and autoradiography. The bands represent full-length siRNA molecules indicating that PEI complexation leads to the efficient protection of siRNAs while noncomplexed siRNAs are rapidly degraded. (b,c) In vivo delivery of intact siRNAs upon PEI complexation. [³²P]-labeled siRNAs, complexed (+) or not complexed (−) with PEI, were injected IP into mice bearing subcutaneous SKOV-3 ovarian carcinoma cell tumor xenografts, and after 30 min (b) or 4 h (b) total RNA from various organ and tissue homogenates was prepared and subjected to agarose gel electrophoresis prior to blotting and autoradiography. The bands represent intact [³²P]-labeled siRNA molecules which for several hours are mainly found in tumor and muscle as well as in liver and, time-dependently, in kidney. Only little siRNA amounts are detected in the lung and traces in the brain.

Figure 3

Protection and in vivo delivery of siRNAs upon PEI complexation. In [70] (a) in vitro protection of siRNAs against nucleolytic degradation. [³²P] end-labeled siRNAs, complexed (upper panel) or not complexed (lower panel) with PEI, were subjected to treatment with 1 % fetal calf serum at 37^°C. At the time points indicated, the samples were analysed by agarose gel electrophoresis, blotting, and autoradiography. The bands represent full-length siRNA molecules indicating that PEI complexation leads to the efficient protection of siRNAs while noncomplexed siRNAs are rapidly degraded. (b,c) In vivo delivery of intact siRNAs upon PEI complexation. [³²P]-labeled siRNAs, complexed (+) or not complexed (−) with PEI, were injected IP into mice bearing subcutaneous SKOV-3 ovarian carcinoma cell tumor xenografts, and after 30 min (b) or 4 h (b) total RNA from various organ and tissue homogenates was prepared and subjected to agarose gel electrophoresis prior to blotting and autoradiography. The bands represent intact [³²P]-labeled siRNA molecules which for several hours are mainly found in tumor and muscle as well as in liver and, time-dependently, in kidney. Only little siRNA amounts are detected in the lung and traces in the brain.

Figure 4

Systemic treatment of mice with PEI-complexed HER-2-specific siRNAs leads to reduced growth of subcutaneous SKOV-3 tumor xenografts due to decreased HER-2 expression. In [70] athymic nude mice bearing subcutaneous tumor xenografts were injected IP with 0.6 nmoles HER-2-specific naked (open circles) or PEI-complexed (closed circles) siRNAs 2–3 times per week and tumor sizes were evaluated daily from the product of the perpendicular diameters of the tumors. Mean +/− standard error of the mean (SEM) is depicted and Student's unpaired t test was used for comparisons between data sets (**P < .03, ***P < .01). Differences in tumor growth reach significance at day 5 indicating the antitumoral effects of the PEI-complexed HER-2-specific siRNAs.