Systemic Delivery of Small RNA Using Lipid Nanoparticles

Abstract

Gene silencing mediated by RNA interference (RNAi) is expected to have a beneficial impact on the treatment of many diseases because of its potency, selectivity and versatility. To maximize the potential of RNAi effectors such as small interfering RNA and microRNA in clinical therapy, the development of a practical delivery system is required, especially for systemic administration. Recent studies demonstrated that chemical modification of these small RNAs and/or encapsulation of them into lipid nanoparticles is a promising strategy to achieve targeted delivery via systemic administration. In this review article, we introduce recent progress of the research on systemic delivery systems for RNAi therapeutics and consider crucial elements for the design of lipid nanoparticles as a small RNA vector.

1. INTRODUCTION

Double-stranded RNA (dsRNA) induces post-transcriptional gene silencing in a sequence dependent manner, which is referred to as RNA interference (RNAi).¹⁾ A guide strand of dsRNA forms the RNA-induced silencing complex (RISC) with Argonaute and other proteins, and binds to complimentary mRNA.²⁾ Argonaute2, one of the component proteins of the RISC, cleaves complimentary mRNA by endonuclease activity.³⁾ A series of events in RNAi occurs in the cytoplasm, suggesting that RNAi therapy with small RNAs, including small interfering RNA (siRNA) and microRNA (miRNA), have no influence on genome DNA. Because the RISC is recycled and cleaves target mRNA continuously,^4,5) the effect of RNAi is potent and sustainable even at a low dose. Furthermore, small RNAs can induce gene silencing in daughter cells after cell division. RNAi induced by small RNAs uses the endogenous enzymatic system, which is critically different from other small nucleic acid therapeutics such as antisense, aptamer, and decoy. RNAi therapy is expected to have a beneficial impact on the treatment of intractable diseases such as cancer. On the other hand, RNAi therapeutics requires a drug delivery system (DDS) for practical use in common with other gene therapeutics, especially for systemic injection of small RNAs. In general, RNA is immediately degraded by RNase, poorly penetrates through the plasma membrane, and induces interferon responses after systemic injection. Therefore, various ideal delivery systems have been developed for RNAi therapy.⁶⁾ Small RNAs have to be delivered only to the cytoplasm, not to the nucleus, in target cells, which is one of the advantages from the point of view of gene delivery. The versatility of small RNAs as potential therapeutics will expand by the use of advanced DDS technologies.

Recent studies demonstrated that chemical modification of small RNAs and/or encapsulation of them into nanoparticles is a promising strategy to achieve significant therapeutic benefits without adverse side effects. In particular, nanoparticle vectors have been used for the systemic administration of siRNA to patients in clinical trials.^7,8) The next generation of nanoparticle vectors for RNAi therapeutics is now being investigated intensely in basic research. Thus, the research of nanoparticle-mediated delivery of small RNAs is rapidly progressing, because nanoparticles can protect small RNAs from enzymatic degradation, enhance their half-life in blood, and deliver them into target cells in vivo. In addition, delivery systems using nanoparticle vectors are expected to be useful for cancer RNAi therapy because nanoparticles can accumulate in tumors via the enhanced permeability and retention (EPR) effect.⁹⁾

2. LIPID NANOPARTICLES FOR SYSTEMIC DELIVERY OF siRNA

Lipid nanoparticles including liposomes are promising non-viral vectors for small RNA delivery and are well-studied in both basic and clinical studies. Because mRNA and miRNA are delivered between cells by exosomes in the endogenous system,¹⁰⁾ systemic delivery of small RNAs with membrane vesicles is considered to be a reasonable approach. Actually, systemic delivery of siRNA using lipid nanoparticles has already been studied in clinical trials.^7,8) In these clinical trials, all candidates of siRNA medicine for systemic use have been developed by use of DDS technology, and many of them are formulated in lipid nanoparticles. In particular, candidate siRNA medicines for cancer therapy certainly require DDS technology to enter clinical trials.

In 2006, Zimmermann et al. first reported gene silencing by intravenous injection of siRNA encapsulated in stable nucleic acid lipid particles (SNALP) in non-human primates.¹¹⁾ This first generation of SNALP contained the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA) as a key lipid component for siRNA delivery. Systemic administration of apolipoprotein B (ApoB) siRNA (1.0 or 2.5 mg/kg) encapsulated in SNALP to cynomolgus monkeys induced RNAi-mediated gene silencing, resulting in significant reductions in ApoB protein, total cholesterol and low-density lipoprotein levels in the blood. In 2010, Semple et al. performed the design of DLinDMA-based lipids with superior delivery capacity and found 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) as a promising lipid for advanced siRNA delivery.¹²⁾ Systemic administration of transthyretin (TTR) siRNA (0.1 mg/kg) encapsulated in DLin-KC2-DMA-based SNALP to cynomolgus monkeys significantly reduced mRNA levels of TTR in the liver. To date, two clinical trials for liver targeting (SNALP-ApoB and ALN-TTR) and two for tumor-targeting (ALN-VSP and TKM-PLK1) have been initiated using SNALP-based delivery of siRNA.¹³⁾

Atu027 is approximately 100 nm lipoplexes composed of liposomes and siRNA targeting protein kinase N3 (PKN3) and is being studied as an anticancer siRNA agent in phase II clinical trials.^7,14,15) The liposomes of Atu027 are composed of β-L-arginyl-2,3-L-diaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride, diphytanoylphosphatidylethanolamine (DPhyPE), and distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG). Knockdown of the PKN3 gene after systemic administration of Atu027 has been demonstrated in mice, rats, and monkeys. Suppression of tumor growth and lymphatic metastasis by Atu027 has also been demonstrated in orthotopic models of prostate and pancreatic cancers. It was suggested that the inhibitory effect of Atu027 on lymphangiogenesis contributes to its anticancer effects.¹⁵⁾ The safety of Atu027 after systemic injection was confirmed in a phase I clinical trial.⁷⁾ The effectiveness of systemic injection of Atu027 against advanced pancreatic cancer is now being studied by combining Atu027 with the standard chemotherapeutic gemcitabine in a phase II clinical trial.

3. CHEMICAL MODIFICATION OF SIRNA FOR SYSTEMIC ADMINISTRATION

Administration of unmodified small RNAs triggers an innate immune response mediated by a toll-like receptor,¹⁶⁾ which causes toxicity and off-target effects related to immune activation.¹⁷⁾ Judge et al. demonstrated that such immune stimulation can be completely abrogated by the use of 2′-O-methyl-modified siRNA without disrupting gene silencing effects.^18,19) SNALP encapsulating 2′-O-methyl-modified siRNA showed potent gene silencing activity without immune stimulation after intravenous administration in mice. This chemical modification technology using 2′-O-methyl-modified RNA is useful in the design of nanoparticle-mediated siRNA delivery systems because delivery vehicles may facilitate the inflammatory cytokine induction caused by unmodified siRNA.

Soutschek et al. reported that the systemic administration of cholesterol-conjugated siRNA induces RNAi-mediated gene silencing and shows therapeutic activity in mice.²⁰⁾ They demonstrated that cholesterol-conjugated siRNA targeting apoB decreased plasma levels of apoB and total cholesterol after intravenous administration in mice. Wolfrum et al. demonstrated that cholesterol-conjugated siRNA binds to high density lipoprotein, low density lipoprotein, and albumin in the blood, and is transferred to the liver.²¹⁾ Conjugation of cholesterol to siRNA prolongs the half-life of siRNA in the blood after intravenous injection. In addition, we previously found that conjugation of cholesterol to siRNA is useful to stabilize the binding between siRNA and lipid nanoparticles.²²⁾ Because the dissociation of a part of siRNA from delivery vehicles had been observed during preparation in some cases, cholesterol-conjugated siRNA was identified as a means to prevent this undesirable dissociation. For systemic use, we prepared phosphate-tetraethylenepentamine (DCP-TEPA)-based polycation liposomes (TEPA-PCL) carrying cholesterol-conjugated siRNA and modified TEPA-PCL with DSPE-PEG by the post-insertion method.²²⁾ Cholesterol was conjugated to siRNA at the 3′-prime of the sense strand, which did not affect the knockdown efficiency, as previously reported.²⁰⁾ Intravenous injection of cholesterol-conjugated siRNA formulated in PEGylated TEPA-PCL modified with cyclic(Cys-Arg-Gly-Asp-D-Phe) (cRGD) peptide significantly induced RNAi-mediated gene silencing in mice²³⁾ (Fig. 1). As introduced here, modification of siRNA with certain compounds, such as cholesterol, to improve the properties of nanoparticles carrying siRNA could be a useful strategy for its systemic use and applicable to other situations.

Fig. 1. RNAi-Mediated Gene Silencing in Vivo after Systemic Administration²³⁾

Cholesterol-conjugated siRNA (2 mg/kg) formulated in TEPA-PCL modified with PEG5000 and cRGD (cRGD-PEG-PCL) was administered intravenously into C57BL/6 mice that had been previously injected with luciferase-expressing B16F10-luc2 cells (1×10⁵ cells/mouse) via a tail vein. Luminescence from the mice was imaged by an in vivo imaging system 15 min post intraperitoneal injection of 3 mg luciferin. Luciferase activity from tumor-bearing lungs after injection with PBS (○), PEGylated TEPA-PCL/luciferase siRNA (◊), cRGD-PEG-PCL/control siRNA (∆) or cRGD-PEG-PCL/luciferase siRNA (▲) was determined at indicated days. Significant differences from the control group are indicated (* p<0.05).

4. SURFACE MODIFICATION OF NANOPARTICLES FOR SYSTEMIC DELIVERY OF siRNA

The surface properties of nanoparticles strongly affect the pharmacokinetics of nanoparticles encapsulating siRNA after systemic administration.²²⁾ Surface coating with PEG is often applied to improve the half-life of the nanoparticles, which increases the chance for siRNA to reach target tissues. In particular, this long circulating property is crucial for nanoparticles to accumulate in tumors by the EPR effects. Figure 2 shows the biodistribution of PEGylated TEPA-PCL in mice after systemic administration. Both the length and the amount of PEG clearly affected the long circulation property of PEGylated TEPA-PCL. The data suggest that the suitable molecular weight of PEG to endow TEPA-PCL with long circulation is approximately 6000. The length and the amount of PEG suitable for avoiding recognition by the reticuloendothelial system (RES) seems to be dependent on physicochemical properties of nanoparticles such as surface charge and size. In general, PEG2000 is long enough to endow neutral liposomes with long circulation. Our studies suggest that relatively long PEG chains are required to improve the biodistribution of positively charged nanoparticles in some cases.

Fig. 2. Biodistribution of PEGylated TEPA-PCL in Mice after Intravenous Injection²²⁾

TEPA-PCL labeled with [³H] cholesteryl hexadecyl ether (74 kBq/mouse) were modified with DSPE-PEG2000 or DSPE-PEG6000. The percent molar ratio of DSPE-PEG to total lipids was 10 or 20%. BALB/c mice were intravenously injected with [³H]-labeled TEPA-PCL or PEGylated TEPA-PCL via a tail vein. Three hours after the injection, the radioactivity in each organ was determined with a liquid scintillation counter. Data are presented as % dose per tissue. Significant differences from the TEPA-PCL group are indicated (* p<0.05, ** p<0.01, *** p<0.001).

On the other hand, a steric barrier formed by PEGylation inhibits the association of nanoparticles with cells.²³⁾ In fact, PEGylation of TEPA-PCL attenuated the cellular uptake and gene silencing effects of siRNA formulated in TEPA-PCL. Therefore, we developed PEGylated TEPA-PCL modified with Ala-Pro-Arg-Pro-Gly or cRGD peptides for the efficient delivery of siRNA. Targeted delivery of siRNA using these vectors showed significant gene silencing and therapeutic efficacy in vivo after systemic administration²⁴⁾ (Fig. 3). As briefly reviewed here, surface modification of PEGylated nanoparticles with certain ligands, such as peptides and antibodies, is recommended for the efficient delivery of siRNA into target cells. Although the first generation of nanoparticles encapsulating siRNA tested in clinical trials are not modified with such ligands, PEGylated nanoparticles modified with appropriate targeting ligands may be tested as advanced vectors for RNAi medicine.

Fig. 3. Therapeutic Effect of siRNA Delivered by Targeted Nanoparticles in Tumor-Bearing Mice²⁴⁾

Cholesterol-conjugated cocktail siRNA targeting c-myc, MDM2, and VEGF (1 : 1 : 1) was formulated in PEGylated TEPA-PCL (siCocktail/PEG) or cRGD-PEG-PCL (siCocktail/cRGD). Cholesterol-conjugated control siRNA was also formulated in cRGD-PEG-PCL (siControl/cRGD). C57BL/6 mice were injected with B16F10-luc2 cells (1×10⁵ cells/mouse) via a tail vein. Each sample (2 mg/kg as siRNA) or PBS was administered intravenously to these mice at 12, 15, and 18 d after the inoculation. Lung weight was determined on day 21 post tumor injection. * indicates p<0.05 vs. control (PBS); ## indicates p<0.01 vs. siControl/cRGD.

5. EXOSOME AS A VECTOR FOR RNAI EFFECTORS

Exosomes released from various kinds of cells are a type of membrane vesicle with a diameter of approximately 50–90 nm. A transmission electron microscopy (TEM) image clearly shows that exosomes are composed of a lipid bilayer.²⁵⁾ The exosomal membrane consists mainly of sphingomyelins, glycerophospholipids and cholesterol.²⁶⁾ The morphology and size of exosomes observed by TEM are similar to those of some kinds of lipid nanoparticles prepared for systemic gene delivery.²⁷⁾ Preparation of exosome mimetics, such as liposomes containing crucial components of natural exosomes, is an interesting approach for drug and gene delivery.²⁸⁾

Besides lipids, exosomes are also enriched in proteins and nucleotides.²⁵⁾ In 2007, Valadi et al. demonstrated that exosomes secreted from certain cells deliver mRNA and miRNA to other cells for intercellular communication.¹⁰⁾ This finding has significant impact and greatly affects the research on delivery systems of small RNAs. Since exosomes were demonstrated to be an endogenous vector of RNA in the body, they have become a central focus as a non-immunogenic vector for RNAi therapeutics.

Alvarez-Erviti et al. generated targeted exosomes presenting neuron-specific RVG peptides in dendritic cells by a genetic engineering procedure, and encapsulated GAPDH siRNA in purified exosomes by electroporation.²⁹⁾ After mice had been intravenously injected with these exosomes via the tail vein, specific gene silencing was observed in the brain.²⁹⁾ In contrast, such gene silencing was not observed in the liver and other organs, suggesting that these exosomes specifically delivered siRNA to the brain after systemic injection. Wahlgren et al. demonstrated that plasma exosomes derived from humans can deliver siRNA to human mononuclear blood cells.³⁰⁾ This study also used electroporation to encapsulate exogenous siRNA into the exosomes. Plasma exosomes effectively delivered mitogen-activated protein kinase (MAPK)-1 siRNA into the target blood cells and induced selective gene silencing. These data suggest that exosomes isolated from the patient’s body fluids can be modified by the insertion of therapeutic siRNA in vitro and subsequently transferred back to the same patient.

Although the use of exosomes as a delivery vector of siRNA is an ideal strategy for RNAi therapy, large-scale preparation and quality control of exosome vectors remains a future task. Another aspect of future research in exosomes is understanding how exosomes deliver RNA to target cells, which is directly informative for DDS research.²⁵⁾ Exosomes are an excellent model to design artificial siRNA vectors such as lipid nanoparticles. Preparation of exosome-like lipid nanoparticles is considered to be a reasonable strategy for the systemic delivery of RNAi effectors.

6. CONCLUSION AND FUTURE PERSPECTIVES

RNAi effectors such as siRNA and miRNA are attractive drug candidates because of their potency, selectivity and versatility. On the other hand, the establishment of a delivery system is indispensable to maximize the potential of RNAi effectors in clinical therapy. In this review, we focused on recent progress in the systemic delivery system for RNAi therapeutics. As reviewed here, systemic administration of siRNA can be achieved by nanoparticle vectors and will provide clinical benefits to patients. To accomplish this goal, continuous research efforts to create innovative nanoparticle vectors are needed.

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