Introduction
Development of novel therapeutics is a ceaseless process as public heath becomes more and more valuable in the society. Today, small molecules and monoclonal antibodies are main players in drug development, but RNA therapeutics is foreseen to soon become a breakthrough technology. RNA therapeutics is advantageous when current techniques fail due to inefficiency of the treatment, complicated schemes of therapy, or newly developed resistance to the drug due to mutational escape. The Food and Drug Administration has recognized the importance of this new approach: two oligonucleotide-based drugs are already approved (pegaptanib 1 , 2 and kynamro 3 , 4 ). Fast track status has been awarded to studies testing small interfering RNAs (siRNAs) for viral infections, and efficacy studies are being conducted in nonhuman primates, while the safety studies are performed in humans. This siRNA-based revolutionary approach can soon come to the market, depending on the results of several active Phase III clinical trials. 5 , 6 This review will focus on siRNA delivery by lipid nanoparticles (LNPs) and the current status of clinical trials for these systems. In the first section, we will describe the different components forming the LNPs and discuss the possible routes for their improvement. We will then report current clinical trials using LNP-formulated RNA and the possible outputs for therapy in the near future. Finally, we will present a critical analysis of selected clinical trials, which would reveal the common logic behind target selection, and demonstrate some examples of lessons learned from failed trials.
LNPs for RNA delivery: structure and requirements
During the last 5 years, siRNA, microRNA (miRNA) mimetics, antagomirs, and messenger RNA (mRNA) have made tremendous progress as potential therapeutics. 7 – 10 However, targeted delivery and endosome escape still remain the major challenges for RNA drug development. siRNAs are short (19–21 nucleotide), double-stranded RNAs that enter the natural RNA interference (RNAi) mechanism and degrade complementary mRNAs by the use of complicated protein machinery. As a result, one can reversibly knock down in vivo a protein of interest (80%–90% decrease) at doses of siRNAs approximately 1–2 mg/kg. Antagomirs sterically block miRNAs by the formation of duplexes, which influences posttranscriptional regulation of gene expression. 11 Artificial mRNAs are used for controlled transient expression of proteins in cancer immunotherapeutics and vaccination. 10 Today, there are several efficient systemic delivery strategies for RNAs that are presently in clinical trial: LNPs, N -acetylgalactosamine conjugates, and dynamic polyconjugates (DPCs). 6 , 12 , 13 Also, there are techniques for local delivery or topical application of RNAs. 14 , 15
Delivery systems based on cationic lipids (liposomes and LNPs) are the most commonly used nonviral vectors for RNA delivery, both in academic studies and clinical trials. 16 – 18 The basic principle of LNP formation is self-assembly that is an integral property of smart nanosystems. Hundreds of lipids have been developed to improve transfer efficiency and stability and decrease immune response and renal escape. Since the first iterations of LNPs, lipids have evolved a lot; from the common 1,2-dioleoyl-3-trimethylammonium-propane/1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine combination to more sophisticated structures ( Figure 1 ), they have become much less toxic and are biodegradable and pH sensitive (smart). From a chemical viewpoint, there are two main structural improvements that make the new lipids more efficient vehicles for siRNA: multiply unsaturated alkyl chains promote significant destabilization of intracellular membrane bilayer, 19 while protonation of the ionizable dimethylaminopropyl group only during lisosome maturation improves LNP initial fusion with the membrane. 20 Moreover, lipidoids, or lipid-like materials, were shown to be very efficient carriers for siRNA. For example, C12-200 ( Figure 1 ) is suitable for multiple animal species, such as rodents and nonhuman primates. 21 , 22 One of the main benefits of lipidoids is the decreased amount of helper lipids needed for effective delivery.
By themselves, cationic lipids are inadequate delivery vehicles for siRNA, and therefore, the so-called helper lipids should be added to form the LNP. 23 Among them are fusogenic phospholipids ( Figure 1B ), which enhance the transfection activity of LNPs by destabilizing the lipid bilayer structure in cell membranes, and polyethylene glycol (PEG) lipids ( Figure 1C ), which decrease the immune response by increasing colloidal stability and shielding LNPs from macrophages. 24 However, high surface content of PEG lipids decreases cellular internalization of LNPs by hindering membrane destabilization; so, an optimal proportion should be found. To overcome this problem, one can decrease molar percentage or use modified variants of PEG lipids that weakly anchor to LNPs and are gradually lost during circulation. 25 Another key component of LNP is cholesterol, which stabilizes nanoparticles by filling the gaps between lipids 26 and enhances the activity of cationic lipids. 27 A common scheme of siRNA–LNP formation includes several steps. The first step is the formation of complexes of siRNA with ionizable cationic lipids in 40% ethanol solution with low ionic strength and pH approximately 4. As siRNAs are rather short in comparison to plasmids, they should be truly covered by lipids to achieve stable incorporation into LNP. Then, these nucleating structures are covered by helper lipids, cholesterol, and finally by PEG-lipids that lead to the formation of LNPs with embedded siRNA ( Figure 2 ). Most of the parameters such as siRNA:lipids ratio, concentrations, and temperature should be optimized empirically.
To decrease glomerular filtration, renal clearance, and generation of immune response, one should control the size (optimal 50–100 nm) and surface charge of the LNPs. The size of LNPs is controlled mainly by concentration of the components during mixing and the fraction of PEG lipids contained in the mixture. Work performed on LNPs also revealed that neutral carriers are more efficient during circulation in the blood and penetration in tumor tissues, while cationic carriers are preferable during cellular uptake and endosomal escape. 28 Indeed, first-generation LNPs were formed during formulation by self-assembly and were disrupted in endosomes, which led to the degradation of RNA molecules in the endosomes rather than RNA release in the cytosol. These results generated the development of more complicated pH-sensitive LNPs that change their charge in endosomes, thus improving RNA escape after penetration into the cell. However, even in this case, only 1%–2% of the total siRNA is released into the cytosol. 29 Moreover, approximately 70% of all internalized nanoparticles are rejected outside the cell by recycling. 30 Hence, future small molecules developed to enhance the uptake and endosome release of LNPs can really propel the use of RNA therapeutics. 31 , 32 Another prospective effector for LNPs is melittin, a known pore-forming peptide 33 that was successfully used for RNA delivery directly 34 or as a masked part of the polymer carrier for RNA. 35 Finally, incorporation of metal nanoparticles in LNPs could improve disruption of the endosome membrane under alternating magnetic fields. 36
As we mentioned earlier, the main problem in targeted delivery of RNA, besides the escape from endosomes after cell penetration, is the specific and efficient delivery to the appropriate cell types of the tissue of interest. Attachment of targeting molecules ( N -acetylgalactosamine – a ligand of ASGPR receptor, 24 , 37 hyaluronan that specifically binds the CD44 receptor 38 ) and antibodies 39 – 42 to the nanoparticle is the main concept presently used for directing the LNP to the cell or the tissue of interest.
Native siRNAs are easily degraded by nucleases in biological fluids. So, considerable chemical modification for in vivo applications using siRNAs is a necessity. The most common modifications for siRNA used in LNP delivery are 2′- O -methyl, 2′-fluoro and phosphorothioate ( Figure 3 ). More intricate modifications are used to increase RNA stability in conjugates, as RNA itself is less sterically protected than when enveloped by LNP. 43 In most cases, data obtained on the activity of modified siRNA in vitro are easily translated in vivo; however, in vivo toxicity and siRNA stability (degradation pattern) should be thoroughly studied.
Most of the LNP-formulated siRNAs and mRNAs are administered by intravenous (IV) injection. However, high stability of LNPs allows for subcutaneous administration in the case of delivery to the liver, 44 which significantly simplifies their future application as therapeutics.
Toxicity and immunogenicity of siRNA–LNP in Phase I clinical trials
LNPs are the carriers most often used in clinical trials for siRNA-based therapeutics, not only because of their obvious advantages over other existing delivery vehicles, such as ease of preparation and versatility, but also their biocompatibility, biodegradability, and accumulated record of clinical use. Unfortunately, being synthetic and quite “unnatural”, lipids can be toxic and promote immune response at high doses. 45 These negative effects are significantly lower in the newer versions of lipids and polymers; however, immunogenicity and toxicity of LNPs can still be an obstacle in clinical application, particularly if the required dose is high or if frequent repetitive application is required.
Novel lipids selected by advanced screening 13 , 21 , 46 facilitate clinical trials, but application of similar LNPs does not guarantee the success of novel siRNA therapeutics. For example, the clinical trial for the treatment of hypercholesterolemia using siRNA/LNPs to target ApoB production was terminated due to immunogenicity ( NCT00927459 ). On the contrary, no serious adverse effects were reported in the ALN-PCS02 trial with similar LNP by Alnylam Pharmaceuticals, targeting the proprotein convertase subtilisin/kexin type 9 ( PCSK9 ) transcript ( NCT01437059 ). This recent study is particularly encouraging, as it demonstrates that selection of proper lipid components can overcome earlier reported toxicity, immunogenicity such as the release of tumor necrosis factor-α, interferon-γ, interleukin-6, interleukin-12, and damage to the liver tissue resulting in elevated levels of transaminases (alanine transaminase, aspartate transami-nase), leukopenia, and thrombocytopenia. 47 – 49
We want to emphasize that application of siRNA/LNP in clinical studies should be based on predictive animal models and understanding the physiology. Also, sharing data is a key point for successful translation of LNP from a bench scale into clinical studies.
Principles and examples of RNA target selection for LNP–siRNA therapeutics
Intervention with RNAi is based either on direct loss of function or gain of function. In the first case, RNAi causes downregulation of overproduced proteins that cause pathological changes, for example, reducing the production of angiotensinogen by the liver during preeclampsia. Gain of function of specific protein can be achieved by removal of negative regulatory feedback. Both approaches are currently being tested in clinical trials in three major areas: antiviral approaches, anticancer therapies, and genetic disorders, but the success of the therapy relies on careful target selection. In this section, we will discuss the common logic behind efficient target selection. All data on current clinical trials of siRNA/LNP are summarized in Table 1 .
Application of the RNAi approach to combat viral infections has several specific advantages. First, targeting several gene products simultaneously will reduce the possibility of accumulated mutations and reinfection with the stable form of the virus. Second, reducing the expression of cell surface antigens, such as hepatitis B surface antigen in hepatitis B virus (HBV), diminishes immunosuppression and activates the T-cell response, leading to seroconversion and eventually functional cure. 30 Finally, fast response to new strains of viruses by minimal changes of siRNA sequences can be provided with this technology.
Promising results have been obtained using siRNA-loaded LNPs in treating liver cancer. Existing LNP-based delivery protocols point toward clinically important siRNA targets expressed in the liver. Also, clinical studies in humans demonstrated little adverse effect to LNPs in the liver or any general immune response to modified siRNA. Therefore, the success of siRNA drug development depends mostly on the careful selection of the drug target expressed in the liver.
Most of the studies targeting cancer-associated pathways with an RNAi approach are at the discovery stage and some researchers express doubts about the accessibility of nonhepatic tumors to LNP-formulated RNAs. Increased permeability of the blood vessels for proteins and particles <20 nm unfortunately does not translate into permeability to LNPs. Hence, the efficacy of local delivery in the tumor remains a challenge. Target selection in cancer is also not as straightforward as it may seem to be. Numerous pathways are activated in cancer; however, blocking an individual pathway usually does not lead to death of a tumor. Nonetheless, enormous unmet needs and a reduced threshold for acceptable severity of adverse effects guarantee the interest of pharmaceutical companies in the development of anticancer drugs. Several pharmaceutical companies demonstrated willingness to bring RNAi therapeutics to Phase I/Phase II clinical trials. Currently, these trials provide the much needed hope for aggressive cancers resistant to existing treatment protocols. Continued success depends on the improvement of efficient delivery technologies while maintaining a good safety profile. If successful, this will attract investments needed for Phase III trials.
RNA therapeutics can be used not only for direct inhibition of key genes of interest but also as a helper for small-molecule drugs or monoclonal antibodies. For example, inhibition of Testican-1 by siRNA restores sensitivity to lapatinib in the treatment of human epidermal growth factor receptor 2-positive gastric cancer. 50 Another interesting example is the simultaneous application of siRNA and 5-fluorouracil in hepatic metastasis of colon cancer, which reduced the incidence of liver metastases. 51
Antiviral therapy
Arbutus Biopharma Corporation recently published the efficacious treatment of macaques in the early stages of infection by Ebola (<3 days after infection) using IV LNP injections with siRNA (TKM-Ebola formulation). 52 However, in the later clinical trials, adverse toll-like receptors-mediated immune response presumably provoked by the LNPs was observed in some patients during dose escalation, causing the termination of the studies at Phase I. The adverse effect was seen during initial injections of high doses, but subsequent injections were well tolerated ( NCT02041715 ). Large-scale animal trials are expected to provide solutions to this problem.
Continuous growth of the population infected with HBV and hepatitis C virus urges the development of RNAi-based treatments. Today, a number of efficient small-molecule drugs exist: nucleoside analogs entecavir or tenofovir for HBV and sofosbuvir or simeprevir for hepatitis C virus. However, strict adherence to the prescribed daily/weekly schedule is necessary for the success of the treatment, while RNAi acts for months, which represents a significant improvement in quality of life. Arbutus Biopharma Corporation developed LNP-based siRNAs targeting three different loci of the gene coding for the HBV surface antigen present in chronically infected patients. This preparation is entering a Phase I trial. Targeting this surface antigen is particularly important, as reduction of its expression can lead to ablation of immune suppression and stimulation of T-cell response of the patient. For the treatment of HBV, two Phase II clinical trials led by Arrowhead Research Corporation are presently ongoing, testing the RNAi dynamic conjugate (DPC) ARC-520 in combination with approved nucleoside analogs ( NCT02065336 and NCT02349126 ). In the case of DPC, the duration of the effect is shorter than that achieved with the LNP-based Arbutus formulation, which might be a disadvantage of the otherwise very promising ARC-520. 53 – 55
Cancer
The miRNA miR-34 is a well-known tumor suppressor, which inhibits cell cycle progression and induces cancer cell death. In their Phase I trials and safety and dose-escalation studies, NCT01829971 Mirna Therapeutics used LNPs (Smarticle LNP) for the delivery of MRX34. This treatment has been well tolerated in 52 patients who received two injections per week during 3 weeks, followed by 1 week off, with a maximal tolerated dose of 110 mg/m 2 . Another cohort of patients received daily administration for 5 days with 2 weeks off until disease progression or intolerance. Two patients (one patient with primary hepatocellular carcinoma [HCC], metastatic to the lung, and another patient with melanoma, metastatic to the lymph nodes) with advanced, metastatic stage IV cancer have achieved 30% tumor shrinkage after treatment with MRX34. 56
Another important target for the treatment of cancer is polo-like kinase 1 (PLK1), a protein that is often overex-pressed in cancer and whose inhibition results in reduced cell division. 57 Inhibition of PLK1 expression prevents the tumor cell from completing the cell division, resulting in apoptosis. Arbutus Biopharma Corporation has completed Phase I studies and is currently conducting Phase II trials in patients with gastrointestinal neuroendocrine tumors, adrenocortical carcinoma, and HCC ( NCT01437007 , NCT01262235 , NCT02191878 ). Phase I trials resulted in a dose-dependent effect and achieved stable disease and partial response in five out of six patients, in addition to a 19.3% reduction in tumor size. 58 This disease model is ideal for LNPs because of the natural biodistribution of the particles in the neuroendocrine tumors. This makes the adrenal cortex and similar structures possible candidates for LNPs. Arbutus Biopharma Corporation is currently evaluating TKM-PLK1 in an expansion cohort with 20 patients who have HCC. This study is designed to evaluate TKM-PLK1’s safety and efficacy in treating HCC patients.
MYC is one of the first discovered classical oncogenes essential for the growth of many tumors, including HCC. 59 Dicerna is presently conducting Phase I studies with DCR-MYC, a Dicer substrate-based RNAi targeting the MYC oncogene, in combination with proprietary EnCore Dicerna’s liposomal delivery technology, 60 in 72 patients with various cancers, including both solid and hematological cancers, and particularly HCC ( NCT02314052 , NCT02110563 ). Patients undergo IV administration once a week for 2 weeks, followed by a week of rest with the potential for follow-up administration. Tumor penetration is the major issue with the EnCore technology, and this is addressed in the clinical trial.
EphA2 is a cell surface receptor tyrosine kinase involved in neuronal cell migration during the development of embryo and is essentially absent in adult tissues. 61 Its overexpression in cancer is associated with poor clinical outcome. 62 EphA2 knockdown in mice reduced tumorigenicity in breast and pancreatic cancer 63 and resulted in reduced tumor growth in a mouse model of ovarian cancer when injected twice a week during 3 weeks and combined with chemotherapeutics. MD Anderson Cancer Center is conducting a Phase I study involving 40 patients with advanced solid tumors, undergoing IV-administration of 1,2-dioleoyl- sn -glycero-3-phosphatidylcholine neutral liposomes loaded with EphA2 siRNA twice weekly for 3 weeks ( NCT01591356 ). 61 Antineoplastic activity was reported, associated with reduced transcription and translation of EphA2.
Noncancer diseases
Experiments with knockdown of PCSK9 give an excellent example of the gain-of-function approach. 64 Selection of PCSK9 was prompted by clinical cases of genetic disorders with gain of function of this protease causing hypercholesterolemia or, on the contrary, loss-of-function mutations resulting in very low levels of plasma cholesterol. It was further revealed that PCSK9 activity has a strong influence on cholesterol levels through degradation of major hepatocyte receptors or low density lipoprotein (LDL) clearing receptors in hepatocytes. PCSK9 binds hepatocyte LDL clearing receptors both intracellularly and extracellularly, leading to their lysosomal degradation. 65 , 66 Patients treated with siRNA against PCSK9 formulated in LNPs had lower LDL-C levels by 60% in the absence of statins, an effect that was sustained for more than 3 months with a single dose of 10 mg/kg ( NCT02314442 ). This success story can be considered the first published clinical data proving the efficacy of a new class of well-tolerated drugs. LDL levels can potentially be effectively controlled by other classes of drugs such as statins, fibrins, bile acid binding resins, niacin, and cholesterol adsorption inhibitors. Moreover, monoclonal antibodies against PCSK9 have also been found to be almost as effective as siRNA. 67 However, there is a strong opinion that RNAi-based drugs will find a niche even in this competitive market, since existing treatments leave a significant number of patients with poorly controlled LDL levels, even at the highest tolerated doses of existing drugs.
Other tricky targets are apolipoprotein C3 (apoC3) in hypertriglyceridemia and angiopoietin-like 3 protein (ANGPTL3) in the genetic forms of mixed hyperlipidemia and severe hypertriglyceridemia. 68 The strategy for the selection of apoC-III for RNAi targeting resembles that implemented in the selection of PCSK9. ApoC-III is located on the surface of triglyceride-rich lipoproteins, including very low density lipoprotein (VLDL), chylomicrons, and their remnants. In two genetic studies, subjects with loss-of-function mutations in the apoC-III gene presented 39%–44% lower levels of triglycerides and ~40% lower risk of coronary heart disease. 69 Patients with a dysfunctional apoC-III have lower cardiovascular risk. Knocking down apoC-III using antisense oligonucleotides reduced apoC-III and triglycerides but increased the “good” facilitating effect of high density lipoprotein-cholesterol, without inducing hepatic steatosis. 70 Genetic studies also pointed out the ANGPTL3 gene as a potential target for RNAi intervention. Mutations in this gene cause familial hypobetalipoproteinemia type 2, 65 and ANGPTL3-deficient patients feature an insulin-sensitive phenotype with lower plasma insulin and glucose levels. This gene encodes a family of secreted proteins expressed predominantly in the liver. Once translated into protein, it is further processed into an N-terminal peptide and a C-terminal fibrinogen chain. The N-terminal chain is a regulator of lipid metabolism and lipoprotein secretion, while the C-terminal chain may be involved in angiogenesis. Silencing ANGPTL3 in cells improves glucose uptake and shifts the secretion of VLDL from VLDL1 to poor VLDL2 during insulin stimulation. 71
Primary hyperoxaluria type I (PH1) is a rare recessive disorder (5 per 1 million births) caused by a mutation in the alanine glyoxylate aminotransferase (AGT) protein, which is encoded by the AGXT gene and is involved in breakdown of hydroxyproline. AGT deficiency causes buildup of oxalate, the metabolite that characterizes PH1 and causes irreversible kidney damage. Ultimate treatment of PH1 requires both liver and kidney transplant. The strategy of Dicerna was to inhibit the liver enzyme glycolate oxidase (GO), which is responsible for converting glycolate into glyoxylate, the precursor of oxalate. LNP RNAi inhibition of GO in a mouse knockout model of PH1 has nearly normalized oxalate levels. In primates, 85% reduction of GO activity was achieved with 0.3 mg/kg of siRNA, an encouraging result for future Phase I clinical trials. At present, there are no small-molecule inhibitors of GO, and due to the orphan status of the disease, such drugs are not expected to appear in the near future. Hence, RNAi therapy gives a real opportunity for the treatment of this rare disease.
Finally, there are targets that present direct pathology-causing proteins. For example, transthyretin (TTR) is implicated in a rare genetic disease caused by amyloidosis of TTR (ATTR). 72 , 73 A mutated variant of this protein causes amyloid deposition in nerve endings and in the heart. However, it is synthesized by the liver and, hence, is an accessible target for siRNA. Therefore, choosing TTR as a drug target is very straightforward. In patients, LNP formulations ALN-TTR01 and ALN-TTR02 suppressed the production of both mutant and nonmutant forms of TTR, establishing proof of concept for RNAi therapy targeting mRNA transcribed from a disease-causing gene ( NCT01148953 and NCT01559077 ). This project is now in Phase III clinical trials, the most advanced stage for an LNP-based therapy ( NCT01960348 ). The trials, led by Alnylam Pharmaceuticals, will serve to evaluate efficacy and safety, with a goal of achieving improved neuropathy impairment scores over placebo with 0.3 mg/kg administration of patisiran every 3 weeks for 18 months in up to 200 patients ( NCT01960348 ). 46 This project is in the discovery stage; however, if successful, it can provide a cure for preeclampsia, a disease with unmet efficacious treatment options. 74
Conclusion
It can be concluded that the present techniques of RNA delivery using LNPs guarantee effective knockdown of any RNA target in the liver. Moreover, there are minimal adverse effects related to the delivery vehicle or the siRNAs themselves. Therefore, the success of siRNA-based drug development in the liver depends mostly on proper target selection. The last 3 years were extremely productive in translation of fundamental studies and preclinical research of RNAi-based therapies into clinical trials. Research in the fields of cancer and antiviral therapies demonstrated the safety and efficacy of an approach based on LNP delivery of siRNA in Phase I/II clinical trials, paving the road for RNAi therapies for the treatment of genetic and metabolic diseases. Alnylam Pharmaceuticals’ PCSK9 studies have demonstrated a competitive edge over existing small molecules and biological intervention in the treatment of hypercholesterolemia. The TTR program is anticipated to receive the first Food and Drug Administration approval for clinical application in the nearest future. Urgent unmet medical needs, particularly in the treatment of patients with viral infections and patients with refractive aggressive cancers, stimulate the development of novel delivery strategies. There is no doubt that we are at the verge of transformation of medical practice by a widespread application of RNAi-based treatment for many human disorders.
Acknowledgments
This work was supported by the Russian Scientific Foundation (grant no 14-34-00017). The authors are grateful to Dominique Leboeuf for critical reading of the manuscript.
Disclosure
The authors report no conflicts of interest in this work.
References
1 Vinores SA Pegaptanib in the treatment of wet, age-related macular degeneration Int J Nanomedicine 2006 1 3 263 268 17717967 PMC2426796
2 U.S. Food and Drug Administration FDA Approves New Drug Treatment for Age-Related Macular Degeneration [press release] 2004 [December 20]. Available from: http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/2004/ucm108385.htm Accessed December 1, 2015
3 Tse MT Regulatory watch: Antisense approval provides boost to the field Nature Rev Drug Disc 2013 12 179
4 U.S. Food and Drug Administration FDA approves new orphan drug Kynamro to treat inherited cholesterol disorder [press release] 2013 [January 29]. Available from: http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm337195.htm Accessed December 1, 2015
5 Zuckerman JE Davis ME Clinical experiences with systemically administered siRNA-based therapeutics in cancer Nat Rev Drug Discov 2015 14 12 843 856 26567702 10.1038/nrd4685
6 Bobbin ML Rossi JJ RNA interference (RNAi)-based therapeutics: delivering on the promise? Annu Rev Pharmacol Toxicol 2016 56 103 122 26738473 10.1146/annurev-pharmtox-010715-103633
7 Lam JK Chow MY Zhang Y Leung SW siRNA versus miRNA as therapeutics for gene silencing Mol Ther Nucleic Acids 2015 4 e252 26372022 10.1038/mtna.2015.23 PMC4877448
8 Chakraborty C Wen ZH Agoramoorthy G Lin CS Therapeutic microRNA delivery strategies with special emphasis on cancer therapy and tumorigenesis: current trends and future challenges Curr Drug Metab 2016 17 5 469 477 26813887 10.2174/1389200217666160126142408
9 Wittrup A Lieberman J Knocking down disease: a progress report on siRNA therapeutics Nat Rev Genet 2015 16 9 543 552 26281785 10.1038/nrg3978 PMC4756474
10 Sahin U Karikó K Türeci Ö mRNA-based therapeutics – developing a new class of drugs Nat Rev Drug Discov 2014 13 10 759 780 25233993 10.1038/nrd4278
11 Kumar V Mahato RI Delivery and targeting of miRNAs for treating liver fibrosis Pharm Res 2015 32 2 341 361 25186440 10.1007/s11095-014-1497-x
12 Kanasty R Dorkin JR Vegas A Anderson D Delivery materials for siRNA therapeutics Nat Mater 2013 12 11 967 977 24150415 10.1038/nmat3765
13 Yin H Kanasty RL Eltoukhy AA Vegas AJ Dorkin JR Anderson DG Non-viral vectors for gene-based therapy Nat Rev Genet 2014 15 8 541 555 25022906 10.1038/nrg3763
14 Sarett SM Nelson CE Duvall CL Technologies for controlled, local delivery of siRNA J Control Release 2015 218 94 113 26476177 10.1016/j.jconrel.2015.09.066 PMC4665980
15 Fujita Y Kuwano K Ochiya T Development of small RNA delivery systems for lung cancer therapy Int J Mol Sci 2015 16 3 5254 5270 25756380 10.3390/ijms16035254 PMC4394474
16 Singh Y Tomar S Khan S Bridging small interfering RNA with giant therapeutic outcomes using nanometric liposomes J Control Release 2015 220 Pt A 368 387 26528900 10.1016/j.jconrel.2015.10.050
17 Leung AK Tam YY Cullis PR Lipid nanoparticles for short interfering RNA delivery Adv Genet 2014 88 71 110 25409604 10.1016/B978-0-12-800148-6.00004-3 PMC7149983
18 Hope MJ Enhancing siRNA delivery by employing lipid nanoparticles Ther Deliv 2014 5 6 663 673 25090280 10.4155/tde.14.37
19 Wan C Allen TM Cullis PR Lipid nanoparticle delivery systems for siRNA-based therapeutics Drug Deliv Transl Res 2014 4 1 74 83 25786618 10.1007/s13346-013-0161-z
20 Maier MA Jayaraman M Matsuda S Biodegradable lipids enabling rapidly eliminated lipid nanoparticles for systemic delivery of RNAi therapeutics Mol Ther 2013 21 8 1570 1578 23799535 10.1038/mt.2013.124 PMC3734658
21 Love KT Mahon KP Levins CG Lipid-like materials for low-dose, in vivo gene silencing Proc Natl Acad Sci U S A 2010 107 5 1864 1869 20080679 10.1073/pnas.0910603106 PMC2804742
22 Novobrantseva TI Borodovsky A Wong J Systemic RNAi-mediated gene silencing in nonhuman primate and rodent myeloid cells Mol Ther Nucleic Acids 2012 1 e4 23344621 10.1038/mtna.2011.3 PMC3381593
23 Cheng X Lee RJ The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery Adv Drug Deliv Rev 2016 99 Pt A 129 137 26900977 10.1016/j.addr.2016.01.022
24 Kumar V Qin J Jiang Y Shielding of lipid nanoparticles for siRNA delivery: impact on physicochemical properties, cytokine induction, and efficacy Mol Ther Nucleic Acids 2014 3 e210 25405467 10.1038/mtna.2014.61 PMC4459547
25 Xia Y Tian J Chen X Effect of surface properties on liposomal siRNA delivery Biomaterials 2016 79 56 68 26695117 10.1016/j.biomaterials.2015.11.056 PMC4698067
26 Dabkowska AP Barlow DJ Hughes AV Campbell RA Quinn PJ Lawrence MJ The effect of neutral helper lipids on the structure of cationic lipid monolayers J R Soc Interface 2012 9 68 548 561 21831895 10.1098/rsif.2011.0356 PMC3262423
27 Tenchov BG MacDonald RC Siegel DP Cubic phases in phosphatidylcholine-cholesterol mixtures: cholesterol as membrane “fusogen” Biophys J 2006 91 7 2508 2516 16829556 10.1529/biophysj.106.083766 PMC1562400
28 Ernsting MJ Murakami M Roy A Li SD Factors controlling the pharmacokinetics, biodistribution and intratumoral penetration of nanoparticles J Control Release 2013 172 3 782 794 24075927 10.1016/j.jconrel.2013.09.013 PMC3891171
29 Gilleron J Querbes W Zeigerer A Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape Nat Biotechnol 2013 31 7 638 646 23792630 10.1038/nbt.2612
30 Sahay G Querbes W Alabi C Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling Nat Biotechnol 2013 31 7 653 658 23792629 10.1038/nbt.2614 PMC3814166
31 Gilleron J Paramasivam P Zeigerer A Identification of siRNA delivery enhancers by a chemical library screen Nucleic Acids Res 2015 43 16 7984 8001 26220182 10.1093/nar/gkv762 PMC4652771
32 Osborn MF Alterman JF Nikan M Guanabenz (Wytensin™) selectively enhances uptake and efficacy of hydrophobically modified siRNAs Nucleic Acids Res 2015 43 18 8664 8672 26400165 10.1093/nar/gkv942 PMC4605330
33 Hou KK Pan H Schlesinger PH Wickline SA A role for peptides in overcoming endosomal entrapment in siRNA delivery – a focus on melittin Biotechnol Adv 2015 33 6 Pt 1 931 940 26025036 10.1016/j.biotechadv.2015.05.005 PMC4540690
34 Hou KK Pan H Ratner L Schlesinger PH Wickline SA Mechanisms of nanoparticle-mediated siRNA transfection by melittin-derived peptides ACS Nano 2013 7 10 8605 8615 24053333 10.1021/nn403311c PMC4013830
35 Wooddell CI Rozema DB Hossbach M Hepatocyte-targeted RNAi therapeutics for the treatment of chronic hepatitis B virus infection Mol Ther 2013 21 5 973 985 23439496 10.1038/mt.2013.31 PMC3666629
36 Golovin YI Gribanovsky SL Golovin DY Towards nanomedicines of the future: Remote magneto-mechanical actuation of nanomedicines by alternating magnetic fields J Control Release 2015 219 43 60 26407671 10.1016/j.jconrel.2015.09.038 PMC4841691
37 Akinc A Querbes W De S Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms Mol Ther 2010 18 7 1357 1364 20461061 10.1038/mt.2010.85 PMC2911264
38 Cohen ZR Ramishetti S Peshes-Yaloz N Localized RNAi therapeutics of chemoresistant grade IV glioma using hyaluronan-grafted lipid-based nanoparticles ACS Nano 2015 9 2 1581 1591 25558928 10.1021/nn506248s
39 McCaskill J Singhania R Burgess M Efficient biodistribution and gene silencing in the lung epithelium via intravenous liposomal delivery of siRNA Mol Ther Nucleic Acids 2013 2 e96 23736774 10.1038/mtna.2013.22 PMC3696903
40 Hajdu P Chimote AA Thompson TH Koo Y Yun Y Conforti L Functionalized liposomes loaded with siRNAs targeting ion channels in effector memory T cells as a potential therapy for autoimmunity Biomaterials 2013 34 38 10249 10257 24075407 10.1016/j.biomaterials.2013.09.019 PMC3900865
41 Ramishetti S Kedmi R Goldsmith M Systemic gene silencing in primary T lymphocytes using targeted lipid nanoparticles ACS Nano 2015 9 7 6706 6716 26042619 10.1021/acsnano.5b02796
42 Weinstein S Toker IA Emmanuel R Harnessing RNAi-based nanomedicines for therapeutic gene silencing in B-cell malignancies Proc Natl Acad Sci U S A 2016 113 1 E16 E22 26699502 10.1073/pnas.1519273113 PMC4711827
43 Ku SH Jo SD Lee YK Kim K Kim SH Chemical and structural modifications of RNAi therapeutics Adv Drug Deliv Rev Epub 2015 11 5 10.1016/j.addr.2015.10.015 26549145
44 Chen S Tam YY Lin PJ Leung AK Tam YK Cullis PR Development of lipid nanoparticle formulations of siRNA for hepatocyte gene silencing following subcutaneous administration J Control Release 2014 196 106 112 25285610 10.1016/j.jconrel.2014.09.025
45 Landesman-Milo D Peer D Transforming nanomedicines from lab scale production to novel clinical modality Bioconjug Chem Epub 2016 1 20 10.1021/acs.bioconjchem.5b00607 26734836
46 Semple SC Akinc A Chen J Rational design of cationic lipids for siRNA delivery Nat Biotechnol 2010 28 2 172 176 20081866 10.1038/nbt.1602
47 Tousignant JD Gates AL Ingram LA Comprehensive analysis of the acute toxicities induced by systemic administration of cationic lipid:plasmid DNA complexes in mice Hum Gene Ther 2000 11 18 2493 2513 11119421 10.1089/10430340050207984
48 Zhang JS Liu F Huang L Implications of pharmacokinetic behavior of lipoplex for its inflammatory toxicity Adv Drug Deliv Rev 2005 57 5 689 698 15757755 10.1016/j.addr.2004.12.004
49 Xue HY Liu S Wong HL Nanotoxicity: a key obstacle to clinical translation of siRNA-based nanomedicine Nanomedicine (Lond) 2014 9 2 295 312 24552562 10.2217/nnm.13.204 PMC4095781
50 Kim HP Han SW Song SH Testican-1-mediated epithelial-mesenchymal transition signaling confers acquired resistance to lapatinib in HER2-positive gastric cancer Oncogene 2014 33 25 3334 3341 23873022 10.1038/onc.2013.285
51 Zhou D Zhang YI Liang D Effect of combination therapy of siRNA targeting growth hormone receptor and 5-fluorouracil in hepatic metastasis of colon cancer Oncol Lett 2015 10 6 3505 3509 26788158 10.3892/ol.2015.3770 PMC4665694
52 Geisbert TW Lee AC Robbins M Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study Lancet 2010 375 9729 1896 1905 20511019 10.1016/S0140-6736(10)60357-1 PMC7138079
53 Yuen MF Chan HL Given BD Hamilton J Schluep T David L Phase II, dose ranging study of ARC-520, a siRNA-based therapeutic, in patients with chronic hepatitis B virus infection Hepatology 2015 62 1385A
54 Yuen MF Chan HL Given BD ARC-520 produces deep and durable knockdown of viral antigens and DNA in a phase II study in patients with chronic hepatitis B Hepatology 2015 62 1385A
55 Gish RG Yuen MF Chan HL Synthetic RNAi triggers and their use in chronic hepatitis B therapies with curative intent Antiviral Res 2015 121 97 108 26129970 10.1016/j.antiviral.2015.06.019
56 Beg MS Brenner A Sachdev J Safety, tolerability, and clinical activity of MRX34, the first-in-class liposomal miR-34 mimic, in patients with advanced solid tumors Mol Cancer Ther 2015 14 C43
57 Liu X Targeting polo-like kinases: a promising therapeutic approach for cancer treatment Transl Oncol 2015 8 3 185 195 26055176 10.1016/j.tranon.2015.03.010 PMC4486469
58 Ramanathan RK Hamburg SI Borad MJ A phase I dose escalation study of TKM-080301, a RNAi therapeutic directed against PLK1, in patients with advanced solid tumors Cancer Res 2013 73 8 Suppl LB-289
59 Abrams M Ganesh S Ying B EnCore-LNP mediated tumor delivery of MYC and CTNNB1 dicer substrate RNAs (DsiRNAs) Mol Cancer Res 2015 13 10 Suppl B20
60 Ganesh S Ying B Koser M Systemic delivery of CTNNB1 dicer-substrate siRNAs (DsiRNAs) leads to efficient oncogene silencing in diverse tumor types of extra hepatic origin Cancer Res 2015 75 15 Suppl 3533
61 Landen CN Jr Chavez-Reyes A Bucana C Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery Cancer Res 2005 65 15 6910 6918 16061675 10.1158/0008-5472.CAN-05-0530
62 Shen H Rodriguez-Aguayo C Xu R Enhancing chemotherapy response with sustained EphA2 silencing using multistage vector delivery Clin Cancer Res 2013 19 7 1806 1815 23386691 10.1158/1078-0432.CCR-12-2764 PMC3618564
63 Ozcan G Ozpolat B Coleman RL Sood AK Lopez-Berestein G Preclinical and clinical development of siRNA-based therapeutics Adv Drug Deliv Rev 2015 87 108 119 25666164 10.1016/j.addr.2015.01.007 PMC4504743
64 Fitzgerald K Frank-Kamenetsky M Shulga-Morskaya S Effect of an RNA interference drug on the synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) and the concentration of serum LDL cholesterol in healthy volunteers: a randomised, single-blind, placebo-controlled, Phase 1 trial Lancet 2014 383 9911 60 68 24094767 10.1016/S0140-6736(13)61914-5 PMC4387547
65 Horton JD Cohen JC Hobbs HH Molecular biology of PCSK9 : its role in LDL metabolism Trends Biochem Sci 2007 32 2 71 77 17215125 10.1016/j.tibs.2006.12.008 PMC2711871
66 Poirier S Mayer G Poupon V Dissection of the endogenous cellular pathways of PCSK9-induced low density lipoprotein receptor degradation: evidence for an intracellular route J Biol Chem 2009 284 42 28856 28864 19635789 10.1074/jbc.M109.037085 PMC2781431
67 Zhang XL Zhu QQ Zhu L Safety and efficacy of anti-PCSK9 antibodies: a meta-analysis of 25 randomized, controlled trials BMC Med 2015 13 123 26099511 10.1186/s12916-015-0358-8 PMC4477483
68 Jørgensen AB Frikke-Schmidt R Nordestgaard BG Tybjærg-Hansen a loss-of-function mutations in APOC3 and risk of ischemic vascular disease N Engl J Med 2014 371 1 32 41 24941082 10.1056/NEJMoa1308027
69 Graham MJ Lee RG Bell TA 3rd Antisense oligonucleotide inhibition of apolipoprotein C-III reduces plasma triglycerides in rodents, nonhuman primates, and humans Circ Res 2013 112 11 1479 1490 23542898 10.1161/CIRCRESAHA.111.300367
70 Wang X Wang D Shan Z Clinical and genetic analysis of a family diagnosed with familial hypobetalipoproteinemia in which the proband was diagnosed with diabetes mellitus Atherosclerosis 2015 239 2 552 556 25733326 10.1016/j.atherosclerosis.2015.02.031
71 Tikka A Soronen J Laurila PP Metso J Ehnholm C Jauhiainen M Silencing of ANGPTL 3 (angiopoietin-like protein 3) in human hepatocytes results in decreased expression of gluconeogenic genes and reduced triacylglycerol-rich VLDL secretion upon insulin stimulation Biosci Rep 2014 34 6 e00160 25495645 10.1042/BSR20140115 PMC4266921
72 Coelho T Adams D Silva A Safety and efficacy of RNAi therapy for transthyretin amyloidosis N Engl J Med 2013 369 9 819 829 23984729 10.1056/NEJMoa1208760
73 Butler JS Chan A Costelha S Preclinical evaluation of RNAi as a treatment for transthyretin-mediated amyloidosis Amyloid 2016 23 2 1 10 27033334 10.3109/13506129.2016.1160882 PMC4898164
74 Haase N Foster D Bercher J RNAi therapeutics targeting human angiotensinogen (hAGT) ameliorate preeclamptic sequelae in an established transgenic rodent model for preeclampsia Hypertension 2014 64 A666