Therapeutic Delivery

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Enhancing siRNA delivery by employing lipid nanoparticles

For several decades extensive research has been conducted into the development of fusogenic lipid nanoparticles (LNPs) capable of introducing large, charged molecules into the cytoplasm of target cells. The majority of this work has focused on cationic LNPs encapsulating nucleic acids ranging from small oligonucleotides to large plasmid constructs thousands of bases long. However, since the introduction of siRNA payloads this quest for a non-viral, intracellular delivery systems has advanced significantly. Of particular importance was the demonstration that LNPs containing ionizable, dialkylamino lipids, enable potent hepatic gene silencing across species including humans. This review focuses on the evolution of this delivery system, summarizes the promising data now emerging from clinical trials and considers future directions for the platform.

Nomenclature: liposomes to lipid nanoparticles Soon after liposomal structures were first described in 1965 it was realized that the biophysical properties of an aqueous space bounded by semipermeable, phospholipid bilayers were well suited for drug delivery [1] . Over the years a wide variety of different lipid-based delivery systems have been investigated, including large multilamellar to small unilamellar liposomes and lipid particles with semi-solid or solid lipid cores, with an equal number of confusing acronyms used to describe them. Consequently, it is now common to use the general term lipid nanoparticle (LNP) to describe lipid-based delivery systems with diameters in the range of 25 to 150 nm, which is considered the most suitable for intravenous administration. The body of literature describing cationic lipids and lipid-based, non-viral particles for the intracellular delivery of polymeric nucleic acids is large and daunting, even for those of us directly involved in the field. However, very few systems have ever been tested in humans and none have achieved the level of success experienced by the class of LNPs described here. Therefore this review is nar-

10.4155/TDE.14.37 © 2014 Future Science Ltd

Michael J Hope*,1 Acuitas Therapeutics, 407-2389 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada *Author for correspondence: Tel.: +1 604 837 9239 [email protected]


rowly focused on LNPs containing ionizable dialkylamino lipids as the cationic lipid component (this class will be referred to as iLNPs). A brief background describing how iLNPs evolved from lipid-based drug delivery research is presented followed by the most recent preclinical and clinical data demonstrating the potent hepatic gene silencing obtained using iLNPs encapsulating siRNA. Quest for enabling delivery technology Over the last two decades a number of clinically successful LNP formulations have achieved regulatory approval [1] . These tend to be formulations that favorably modify pharmacokinetics and pharmacodynamics of conventional, small molecular weight drugs already in clinical use, thus providing an incremental improvement in their therapeutic profile. However, demonstrating small improvements in clinical benefit for regulatory bodies is challenging, usually requiring a prolonged clinical trial process, which has impeded widespread adoption of LNP delivery technology by the pharmaceutical industry. This is despite the fact that many innovative advances have been made to address

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ISSN 2041-5990


Review  Hope

Key Terms iLNPs: Lipid nanoparticles 60–80 nm in diameter composed of phospholipid, cholesterol, PEG-lipid and an ionizable amino lipid with two unsaturated (fluid) hydrophobic chains. Delivery to hepatocytes is maximized when the amino lipid headgroup has a pKa~6.4 Non-bilayer structure: Structure adopted by lipids that reflects their molecular shape. Lipids with a cylindrical shape form bilayers, but cone-shaped lipids adopt nonbilayer structures. An example is the hexagonal HII phase where the lipid headgroups orientate around narrow tubes of water stacked in a hexagonal array. Lipid mixtures containing both cylindrical and cone shapes can also form inverted micelles between membranes. These non-bilayer structures are thought to be intermediates in fusion and also destabilize the normal bilayer structure. Self-assembly: In an ethanol/aqueous solution with acidic pH, the iLNP lipid components spontaneously form iLNPs in a self-assembly process that encapsulates siRNA. Particle formation is driven by the electrostatic attraction between positively charged amino lipid and negatively charged nucleic acid. Once iLNPs have formed the pH is raised for administration.

practical aspects of development in areas such as manufacturing, drug loading and stability [1] . A good example is Marqibo®, a liposomal formulation of the ubiquitous anticancer drug vincristine, better tolerated than free drug [2–4] , which was first developed in the late 1980s [5–7] , but only achieved US FDA approval in 2012 for treatment of acute lymphoblastic leukemia. As a result, researchers in this field strive to identify LNP technology that can do more than make a lookalike of an existing drug. Ideally, the delivery system should be an essential component of drug activity, and thus enable new classes of therapeutics [8] . One area that has been studied extensively from this perspective is the creation of LNPs capable of controlled fusion with target cell membranes in a manner similar to many viruses. Drug-delivery systems that can fuse with cell membranes should enable the intracellular delivery of therapeutic molecules with biophysical properties that prevent their passive diffusion through membranes to access sites of action. Many potential protein and nucleic acid-based drugs fall into this therapeutic category due to their large molecular weight and charge. During the 1980s a better understanding of the biophysics of membrane fusion emerged. It became clear that viruses employ proteins to control their association and subsequent fusion with cells, and many studies were undertaken to incorporate viral fusion proteins into LNPs. However, with the exception of vaccine adjuvants [9] , this approach has proven difficult to translate into vehicles for the systemic delivery of large molecules, mainly because of issues surrounding large-scale manufacturing and immune toxicity.


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The role of lipids in membrane fusion was also under active investigation at this time. Of particular interest was the observation that phosphatidylethanolamine (PE), one of the most ubiquitous mammalian membrane phospholipids, prefers to adopt non-bilayer structures in isolation. An extensive characterization of this lipid resulted in the hypothesis that lipid polymorphism (the shape of the molecular envelope exhibited by lipids when fully hydrated) is integral to the mechanism of membrane fusion and that unsaturated PE may be considered a naturally occurring fusogenic lipid [10] . Briefly, phospholipids that spontaneously form bilayer structures adopt a cylindrical shape, which is compatible with packing into membrane bilayers. PE, on the other hand, exhibits a cone shape and when it is separated from bilayer stabilizing lipids it spontaneously adopts a non-bilayer structure called the hexagonal HII phase (Figure 1) . Model membranes containing mixtures of bilayer lipids and PE can be manipulated to fuse by inducing subtle changes to lipid shapes by altering pH and ionic environment [10] ; they also form inverted micelles, which are intermediate structures thought to form as two bilayers undergo fusion. Consequently, PE was incorporated into LNP drug-delivery systems in an attempt to provide them with a fusogenic capacity; however, the inherent instability of model membranes containing PE proved difficult to control [10] . Cationic lipids enable nucleic acid delivery into cells A significant advance in the quest for fusogenic delivery systems occurred when Felgner and colleagues [11,12] , demonstrated that LNPs composed of cationic lipids and unsaturated PE could transfect cells in vitro with plasmid DNA. This work showed for the first time that non-viral, protein-free, lipid vehicles could deliver large molecular weight and highly charged molecules through membrane barriers and release them inside the cell. Key here is the cationic lipid component, which plays multiple roles in both the formulation and delivery processes. First, the positive charge provides a well hydrated lipid headgroup that helps stabilize the cone shape of the fusogenic ‘helper’ lipid (unsaturated PE) in a bilayer configuration. Second, the cationic lipid binds to negatively charged phosphates of the nucleic acid backbone, which not only condenses plasmid DNA but also orients the hydrophobic lipid chains outwards, essentially turning the nucleic acid into a hydrophobic particle. Remaining lipids organize with their headgroups facing the aqueous medium, thus providing a hydrophilic surface to stabilize the hydrophobic lipid–DNA complex in aqueous suspension; the resulting lipid–plasmid aggregates are often referred to as lipoplexes [13] .

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Enhancing siRNA delivery by employing lipid nanoparticles 

Amino lipid

Anionic phospholipid

Ion pair

Endosome acidifcation

Cylindrical shape supports bilayer structure





Cone shape disrupts bilayer structure

Hexagonal HII

Figure 1. Lipid shape and the proposed mechanism of action for membrane disruptive effects of ionizable amino lipids. In isolation, amino lipids and endosomal membrane anionic lipids such as phosphatidylserine adopt a cylindrical molecular shape, which is compatible with packing in a bilayer configuration. However, protonated (cationic) amino lipids interact with anionic lipids to form ion pairs where the cross-sectional area of the combined headgroup is less than that swept out by the hydrocarbon tails. The ion pair therefore adopts a molecular ‘cone’ shape, which promotes the formation of inverted, non-bilayer phases such as the hexagonal HII phase illustrated here. Inverted phases do not support bilayer structure and are associated with membrane fusion and membrane disruption. Reproduced with permission from [43] .

Residual positive charge on the external surface of lipoplexes promotes their association with the negative surfaces of cells and induces endocytosis. The presence of unsaturated PE and cationic lipid supports fusion and/or lipid mixing with both plasma and endosomal membranes, resulting in destabilization and release of DNA into the cell cytoplasm [14] . Lipoplex transfection can be highly efficient and is well tolerated by cells in vitro, making formulations such as Lipofectamine® successful transfection reagents. Unfortunately, the transfection efficiency observed in vitro does not translate to in vivo. The positive surface charge and inherent instability of particles containing unsaturated PE produces unacceptable levels of toxicity in animal models and transgene expression observed in vivo rarely achieved therapeutically significant levels. However, the basic principles of particle self-assembly and endosome disruption still apply when considering iLNP technology in clinical development today, but significant refinements were introduced to address the negative aspects of lipoplexes.

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Polyethylene glycol lipids & ionizable amino lipids Polyethylene glycol lipids (PEG-lipids) were incorporated into LNPs to provide a hydrophilic shield at the particle surface [15] . PEG-lipids are most commonly used in LNP drug-delivery systems to increase the circulation lifetimes of particles, the PEG headgroup acts as a steric barrier that inhibits binding of plasma proteins, including opsonins, which mark LNPs for rapid clearance by phagocytes [16,17] . The same shielding principle reduces the tendency for positively charged LNPs to associate with negatively charged plasma components and cell surfaces, which contribute to toxicity. However, the PEG shield also plays an important role in the process of self-assembly during formulation by limiting the size of nascent particles that are formed as lipids coalesce around the nucleic acid polymers. In the absence of PEG-lipids, particle–particle crosslinking and aggregation can grow nucleic acid-containing LNPs to sizes unsuitable for intravenous delivery [18] . Later modifications include shortening the hydrophobic anchor of PEG-lipids [19] , which enables the


Review  Hope

Key Term RNA-induced silencing complex: Cytoplasmic, multiprotein complex that incorporates one strand of a siRNA, which it uses as a template for recognizing and then cleaving complementary mRNA. RISC is part of an important natural process whereby gene expression can be regulated by RNA.

shield to rapidly dissociate from LNPs in circulation so that it does not interfere with subsequent delivery mechanisms [20–22] . Another modification to prove highly significant was the use of ionizable amino lipids rather than the permanently charged cationic lipids common at the time [23–25] . For example, 1,2-dioleyl-N,N-dimethyl3-aminopropane (often referenced using the acronym DODAP), was first synthesized to provide a means of controlling LNP fusion by pH [26,27] . When in a bilayer the ionizable amino propane headgroup exhibits an apparent acid dissociation constant (pK a) that is approximately 7, which is the pH at which 50% of the lipid molecules are protonated and therefore positively charged. The charge increases exponentially as the pH becomes more acidic, similarly, charge decreases as the pH rises. This simple biophysical property means that particle self-assembly can proceed at acidic pH, when positive charge and electrostatic interactions with nucleic acid are maximized. However, once particles have been formed and nucleic acid encapsulated, pH can be raised to reduce the positive surface charge of particles prior to injection [18,28] . Various iterations of iLNPs encapsulating antisense deoxyoligonucleotides (ODN) were investigated in vivo with mixed success [29–32] . One problem to emerge was the realization that many biological responses, used as surrogate markers to demonstrate successful cytoplasmic delivery in animal models, were in fact the result of non-specific immune stimulation rather than the specific knockdown of gene expression the nucleic acid payloads were designed to elicit. For example, iLNPs encapsulating DNA sequences that also contain immunostimulatory CpG motifs activate Toll-like receptor 9 (TLR-9) in macrophages and other antigen-presenting cells of the innate immune system [33] . Shortly after intravenous administration, these sequences induce a complex cascade of cytokines, which have diverse biological consequences, including the activation of natural killer cells that can induce excellent tumor regression [34,35] . Such immune effects are easily interpreted as being the result of specific gene knockdown or gene expression. Interpretation is further confounded by the fact that the immunostimulatory effects are greatly enhanced by encapsulation because particulate delivery systems naturally target antigen-presenting cells where TLR-9


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receptors are located inside endosomes [34–37] . This latter point is important to note, because it means nonfusogenic delivery systems containing TLR-9 ligands do not have to escape the endosomal compartment to trigger immune responses [38] . The resulting confusion surrounding cause and effect of biological data further complicated the development of iLNPs capable of unambiguous cytoplasmic delivery in vivo. Hepatic gene silencing RNA interference is a highly potent, naturally occurring mechanism to control gene expression. Synthetic siRNA molecules can induce RNA interference, but only if they access the cytoplasm of target cells. Once in the cytoplasm, however, siRNA molecules readily load into the RNA-induced silencing complex (RISC) where they direct sequence-specific cleavage of target mRNA. The result is a profound reduction in target mRNA and corresponding protein, which can last for many days after a single dose. RNA oligonucleotides can also activate TLRs and the immune system, but lessons learned from ODN and plasmid applications meant that immune stimulatory sequences were reduced or modified early in siRNA development [39] . Consequently, siRNA became the ideal tool with which to develop iLNPs capable of cytoplasmic delivery. In 2006, a seminal study was published by Zimmermann et al. demonstrating potent in vivo hepatic gene silencing in rodents and non-human primates using siRNA iLNPs [40] . The siRNA was administered intravenously employing iLNPs containing 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA) as the cationic component [41] . This work provided unambiguous evidence that cytoplasmic delivery of siRNA to tissues in vivo by iLNPs was not only possible, but met criteria of potency, duration of response and tolerability sufficient to support clinical development. A single dose of 2.5 mg siRNA/kg reduced target apolipoprotein B (ApoB) mRNA by >90% 48 h after administration and the expected RISC cleavage products were identified in liver tissue. Moreover, reductions in ApoB protein, serum cholesterol and lowdensity lipoprotein (LDL) were measured within 24 h of administration and lasted for 11 days. Critically, this work also demonstrated that simple in vivo screening assays could now be used to define the structure activity relationship (SAR) for iLNPs, and particularly the dialkylamino lipid. Proposed mechanism of delivery The extensive history underpinning this technology meant that a detailed working hypothesis was already in place to act as a guide for the rational design of novel amino lipids. At the core of the proposed mechanism of

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Enhancing siRNA delivery by employing lipid nanoparticles 

delivery is the ability of the amino lipid, when positively charged, to form an ion pair with negatively charged membrane phospholipids [42] ; the resulting charge neutralization decreases the area of both headgroups, causing the lipid pair to adopt a cone shape (Figure 1) . As discussed earlier, this is the molecular geometry adopted by PE, the fusogenic ‘helper’ lipid used in earlier iterations of the delivery platform; therefore, when positively charged, the amino lipid alone can disrupt membrane structure. For amino lipids with a pK a ~7 the surface positive charge of the LNP is low at physiological pH, which minimizes interactions with negatively charged proteins and cell surfaces. However, when LNPs are endocytosed the surface positive charge increases exponentially as the endosomal compartment acidifies subsequently ion-pairs form between amino lipid and negatively charged membrane lipids such as phosphatidylserine. The resulting non-bilayer structures disrupt the endosomal membrane and siRNA gains access to the cytoplasm where it can load into RISC [43] . Despite this well-defined hypothesis it was not clear why such potent gene silencing was observed in hepatocytes, but not in other cells or tissues. This was resolved in 2010 with the discovery that hepatocytes take up dialkylamino iLNPs by apolipoprotein E (ApoE)dependent, receptor-mediated endocytosis [20] . Ionizable LNP of this type are essentially inactive in ApoE knockout mice, but gene silencing is restored when the carriers are mixed with exogenous ApoE prior to administration. ApoE is an important factor in lipoprotein metabolism and is required for liver uptake of very low density lipoprotein (VLDL) and chylomicron remnants [44] , formed by the action of lipases, which hydrolyze the triglyceride core of VLDL and chylomicrons to release fatty acids. As hydrolysis proceeds the lipoproteins become smaller and enriched in ApoE, which targets them for removal by hepatocytes. It is interesting to note, therefore, the similarities between iLNPs and lipoproteins, for example, their diameter of 60–80 nm falls within the size range of VLDL and chylomicron remnants [45] . Lipoproteins also contain a solid lipid core of neutral lipid and recent structural analyses combined with molecular modeling indicate that siRNA LNPs also exhibit an electron-dense, semi-solid core, which appears to consist of an array of nanostructured aqueous compartments, some of which contain siRNA [46] . Moreover, when LNPs are viewed by cryoelectron microscopy, they exhibit a morphology that closely resembles that observed for lipoproteins [46–48] . Optimization for hepatic gene silencing ApoE-dependent uptake is a good demonstration of why lipid-based, drug-delivery systems must be optimized in vivo and not in vitro, as interactions between

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LNPs, plasma components such as proteins, lipoproteins and different cellular surfaces are key participants in the overall mechanism of delivery and greatly impact pharmacokinetics and pharmacodynamics [49,50] . Despite the excellent activity observed using iLNPs containing DLinDMA, this amino lipid evolved from in vitro studies and its structure was not optimized for in vivo hepatic gene silencing [41] . The success of ApoB knockdown prompted the use of a simple in vivo screening assay to conduct a comprehensive SAR analysis of DLinDMA and its analogues [43] . The model employed siRNA targeting murine clotting factor VII (FVII) mRNA; the FVII gene is only expressed in hepatocytes and the protein product is secreted into the blood where its plasma concentration can be readily measured 24–48 h post-administration [51] . Hundreds of amino lipids were synthesized and tested in the same basic LNP structure so that differences in activity could be attributed only to the amino lipid component. The potency of each amino lipid was compared by measuring its median effective dose (ED50) for FVII silencing, estimated from plots of siRNA dose versus plasma FVII concentration [43,52] . Active molecular moieties were identified then modified and incorporated into new lipids for testing, enabling the rational design of progressively more potent molecules. The most significant new SAR discovered relates to the ionization properties of the amino lipid headgroup. Prior to these studies a pK a ~7 or lower was generally considered sufficient to maximize tolerability and other molecular factors were most likely more important determinants of potency in vivo [43] . However, as the number of novel amino lipids tested increased a pattern emerged showing maximum activity was associated with amino lipids that exhibit a surprisingly narrow range of pK a’s, even for lipids with diverse headgroup structures [52] . The optimal apparent pK a was estimated to be 6.4 (measured in situ using fully formulated iLNPs) and the activity of amino lipids with pK a’s above or below this value decreases dramatically [43,52] . The dominance of pK a in the mechanism of delivery is further illustrated by the fact that low activity amino lipids with pK a’s above and below the optimal value can be mixed to obtain iLNPs with an average optimal pK a, which now exhibit high activity [52] . Recent studies also confirm earlier conclusions that alkyl chains must be fluid e.g. typically 18 carbons long with one or two double bonds per chain [42,41] . The requirement for lipid fluidity in membrane fusion is well known and consistent with the role of lipid shape in the proposed mechanism of delivery (Figure 1), as the cross sectional area swept by the chains increases with unsaturation thus exaggerating the cone geom-


Review  Hope etry. It was also found that the interfacial linker that connects the hydrophobic chains to the headgroup must be resistant to hydrolysis in order to maximize activity. For example, if the non-hydrolysable ether linkers found in DLinDMA are substituted by esters (the resulting lipid is often called DODAP in the literature) then hepatic gene silencing activity is greatly reduced, most likely as a result of rapid chain cleavage by lipases following endocytosis [53] . The narrow pK a requirement for maximum activity of iLNPs can also be explained using the mechanism of delivery model (Figures 1 & 2), particularly in light of the ApoE-dependent uptake pathway. ApoE prefers to associate with neutral lipid surfaces [54] , where it undergoes a structural transition to reveal the binding site recognized by receptors on hepatocytes [55,56] . The sharp pK a dependence appears to arise from the need to balance two opposing requirements for activity. In circulation LNPs must exhibit a charge neutral surface to minimize non-specific interactions with plasma components and cells as well as to maximize ApoE binding; therefore a low pK a (7) is ideal. When the pH of the blood and acidified endosomal compartments are taken into account the optimal amino lipid pK a can be calculated to be ~6.4, the same as that determined experimentally [52] . The level of understanding for amino lipid SAR has enabled the rational design and synthesis of lipids that exhibit remarkable potency in vivo. One such lipid is dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA), referred to as MC3 [52] . Using MC3 iLNPs FVII ED50’s have been measured as low as 0.005 mg siRNA/kg in murine models [52] , equivalent to just 0.25 μg siRNA per animal [20] . MC3 formulations encapsulating siRNA therapeutics are currently in Phase II clinical trials for hypercholesterolemia and Phase III trials for transthyretin ameloidosis [57] . Clinical trials Alnylam and Tekmira are two biotechnology companies that currently dominate clinical trials employing iLNPs to deliver siRNA therapeutics. In this section the published clinical results are summarized, but there are a number of other products in early preclinical/clinical development for which information is publicly available through the company websites. As described above, the iLNP platform specifically targets hepatocytes through ApoE receptor-mediated endocytosis; therefore, all the iLNP-based clinical trials todate focus on hepatic gene silencing. Interestingly, the pace at which amino lipid potency has improved over the last 2–3 years has made it challenging to determine


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when to lock down a formulation and commit to clinical development. However, MC3-based iLNPs appear to have established a benchmark performance and Alnylam’s MC3 formulation encapsulating siRNA targeting transthyretin mRNA (ALN-TTR02; recently designated the name Patisiran) is the most advanced with Phase III trials initiated in October 2013. Transthyretin amyloidosis

Circulating transthyretin (TTR) is produced in hepatocytes and genetic variants of the protein are associated with the formation of life-threatening amyloid deposits in various tissues, including heart, kidney and peripheral nerves. Wild-type TTR also accumulates in the amyloid plaques; therefore, even when mutant TTR is resolved by liver transplant, disease progression can still occur. Patisiran silences both mutant and wild-type TTR proteins and preclinical models have shown that reducing plasma concentrations of TTR results in plaque regression [57] . The TTR Phase I study demonstrates the potency of siRNA when coupled with iLNP delivery. In a placebo-controlled, single-dose escalation study in healthy volunteers, the highest dose (0.5 mg siRNA/kg) resulted in a rapid 80% reduction in serum TTR levels by day 4, a 94% reduction at the 10-day nadir, and even after 28 days knockdown was still 77%. Moreover, doses at 0.15 and 0.3 mg/kg were only marginally less active with 10-day nadirs of ~80% knockdown and overall Patisiran was well tolerated [57] . Hypercholesterolemia

Given the specificity of the delivery platform for hepatocytes it is not surprising that hypercholesterolemia has been targeted by siRNA iLNP products. Apolipoprotein B (ApoB) is a primary component of atherogenic LDL and, as discussed earlier, LNP-mediated siRNA knockdown of ApoB decreased LDL cholesterol levels in non-human primates – data that represent a major turning point in the development of siRNA therapeutics. A first-generation DLinDMA-based iLNP formulation encapsulating siRNA targeting ApoB-100 mRNA was taken into the clinic by Tekmira, however, the Phase I trial was halted during dose escalation. Results from a Phase I trial of a second-generation MC3-based iLNP siRNA targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) were recently published by Alnylam [58] . PCSK9 binds to LDL receptors both intracellularly and extracellularly resulting in their degradation, and preclinical models have demonstrated that siRNA knockdown of PCSK9 results in substantial and durable reductions in LDL cholesterol [59] . A rapid, dose-dependent reduction in plasma PCSK9 was observed in healthy volunteers,

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Enhancing siRNA delivery by employing lipid nanoparticles 







LNP with bound ApoE

siRNA payload

Figure 2. Proposed mechanism by which siRNA is delivered to hepatocytes by iLNPs. (A) iLNPs are formed by mixing dialkyl amino lipid, PEG-lipid and structural lipids with siRNA at acidic pH when the amino lipid is positively charged. PEG-lipid provides a hydrophilic PEG shield at the particle surface that limits the growth of nascent particles during the self-assembly process; the target size is typically 60-80 nm. (B) At pH 7.4 iLNPs are essentially uncharged and when they are administered intravenously they rapidly shed the PEG-lipid and bind ApoE. The small size of iLNP enables them to pass through the fenestrated sinusoids of the liver into the space of Disse where they are captured by hepatocyte receptors with ApoE binding sites such as the LDL receptor and LDL receptor related protein and endocytosed. (C) As the endosomal compartment acidifies the amino lipid charge increases and disrupts the endosomal membrane allowing siRNA to escape into the cytoplasm, where it can load into RISC and direct site-specific cleavage of target mRNA. ApoE: Apolipoprotein E; LNP: Lipid nanoparticle; RISC: RNA-induced silencing complex.

with a group mean 70% reduction measured at day 3 for the highest dose of 0.4 mg/kg with an individual maximum knockdown of 84%. The duration of response increased with increasing dose, which was also observed in the TTR trial, and was accompanied by the expected dose-dependent decrease in LDL cholesterol, reaching a mean 40% reduction at the highest dose. Cancer

Alnylam has completed and published a Phase I study for ALN-VSP, a first-generation iLNP containing two encapsulated siRNAs that target kinesin spindle protein (KSP) and vascular endothelial growth factor-A (VEGF), which is being developed for the treatment of primary and secondary liver cancer. This formulation also highlights another exciting aspect of this technology platform, namely the potential to target multiple gene products by encapsulating different siRNA sequences in the same iLNP delivery vehicle [60] . Dosed every 2 weeks, ALN-VSP was well-tolerated and 1 mg/kg was established as the recommended

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dose for Phase II. One patient with endometrial cancer and multiple hepatic metastases experienced a complete response after 40 doses and the patient was in remission when treatment was stopped after 50 doses. Several patients achieved stable disease and siRNAspecific cleavage of target mRNA was observed in tumor biopsy samples. A similar Phase I study has also been completed by Tekmira for TKM-PLK1-001, a first-generation LNP encapsulating siRNA targeting Polo-like kinase I mRNA. The results of this trial have yet to be published, but have been disclosed in company press releases where it is stated that TKM-PLK-001 was generally well-tolerated and that the estimated maximum tolerated dose is expected to be 0.75 mg/kg. Three out of four patients in the Phase I cohort with adrenal cortical carcinoma achieved stable disease and both patients with gastrointestinal neuroendocrine tumors experienced clinical benefit. As a result, Tekmira announced in 2013 that a Phase I/II study of TKM-PLK has been initiated in patients with these cancers.


Review  Hope Antiviral therapies

Encouraging preclinical studies have demonstrated that siRNAs targeting key viral mRNAs significantly reduce viral loads of hepatitis B [61] and can protect animals exposed to lethal doses of filoviruses responsible for hemorrhagic fever in humans [62,63] . Ebola and Marburg filoviruses cause severe morbidity and mortality in humans and NHPs, but there are currently no approved vaccines or therapeutics to treat infections. These viruses also represent a considerable biosecurity concern to governments because of their potential use as biological weapons. The US Department of Defense (Medical Countermeasures) has contracted Tekmira to develop an iLNP formulation containing siRNA targeting Ebola mRNA (TKMEbola). Using a first-generation iLNP encapsulating a mixture of siRNAs targeting multiple Ebola mRNAs, the group reported complete post-exposure protection of infected NHPs [62] . The same group also demonstrated that a siRNA cocktail targeting Marburg mRNAs encapsulated in iLNPs is also highly effective, providing 100% protection to infected guinea pigs [63] . Tekmira announced in January 2014 that a volunteer Phase I study has been initiated using a more potent, second-generation TKM-Ebola iLNP formulation. Conclusion Dialkylamino iLNPs are currently the most advanced siRNA delivery platform for intravenous delivery and have enabled siRNA therapeutics to enter mainstream drug development. The rational design of amino lipids and iLNPs has evolved over more than two decades of research, but development recently accelerated following the introduction of siRNA, which enabled fine tuning of key lipid components to maximize in vivo hepatic gene silencing. The resulting detailed understanding of amino lipid SAR and the role of endogenous ApoE in receptor-mediated uptake of iLNPs by hepatocytes has given rise to an advanced generation of siRNA formulations, which are highly potent and well tolerated in humans. Consequently, a number of siRNA therapeutics employing this delivery technology are in Phase I, II and III clinical trials with several others in late-stage preclinical development, targeting hepatic mRNAs that code for proteins associated with a broad spectrum of diseases. Future perspective Given the remarkable potency and tolerability of the current generation of iLNPs in enabling siRNAinduced hepatic gene silencing, the future looks promising for the continued development of this delivery


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platform. Ironically, one area of interest is to enhance potency even more, this is because recent data indicate only 2–5% of siRNA that enters the endocytic pathway escapes into the cytoplasm [64,65] . Therefore, improvements in endosome disruption and release should significantly boost potency. Achieving this will require not only improvements in endosome disruption but also a better understanding of endosome maturation, trafficking and receptor recycling within the target cell [64] . The ability of iLNPs to utilize ApoE-dependent endocytosis to access hepatocytes at very low intravenous doses clearly demonstrates the power of receptor targeting. For example, iLNP activity can be restored in ApoE knockout mice by incorporating a multivalent N-acetylgalactosamine (GalNAc) lipid into the particle, which targets hepatocyte asialoglycoprotein receptors, enabling iLNPs to bypass ApoEdependent endocytosis [20] . Interestingly, Alnylam has conjugated GalNAc-clusters to chemically modified, nuclease-resistant siRNA molecules, which also knockdown hepatic genes when administered subcutaneously (Alnylam press releases), a route of administration offering advantages over the intravenous route when managing chronic diseases. The success of GalNAc conjugates of siRNA also demonstrates that endosomal escape can occur in the apparent absence of a fusogenic factor and understanding the mechanism should help design better delivery systems across all platforms. Cholesterol conjugates of siRNA also target the liver, but are orders of magnitude less potent than the equivalent siRNA dose encapsulated in iLNPs [40] . However, Arrowhead recently demonstrated that co-administering siRNAcholesterol conjugates with a GalNAc-targeted fusogenic peptide can enhance knockdown 500-fold [66] . Future directions for siRNA delivery will almost certainly include the use of endogenous and/or synthetic ligands to target siRNA and iLNPs to cells and tissues outside the liver. Finally, the use of iLNPs to deliver different nucleic acid therapeutics represents an emerging area of interest. Multiple oligonucleotide therapeutics are in clinical trials, many targeting the liver [67] ; the majority are chemically modified antisense ODN, which are taken up by hepatocytes and do not require a delivery system [68] . However, not all polymeric nucleic acid drugs are taken up by hepatocytes at doses that make them viable drug candidates or can be chemically modified to limit nuclease degradation in circulation. One application that seems set to expand is the use of iLNPs for delivery of mRNA for hepatic protein expression and secretion [69,70] .

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Enhancing siRNA delivery by employing lipid nanoparticles 

Financial & competing interests disclosure The author is an inventor on several patents relating to LNPs, iLNPs and dialkyl amino lipids. He was a co-founder of Tekmira Pharmaceuticals and an employee until 2008, when he left to start Acuitas Therapeutics; he still owns Tekmira shares. Until mid-2012, Acuitas received some funding from Alnylam Pharmaceuticals to develop iLNPs for delivery of siRNA. Acuitas no-longer works with Alnylam or siRNA but now focuses


on the application of iLNPs to deliver other oligonucleotide therapeutics. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Executive summary Quest for enabling delivery technology • Ionizable dialkylamino lipids evolved out of research to develop non-viral, fusogenic delivery vehicles for large molecules such as oligonucleotides.

Hepatic gene silencing • Lipid nanoparticles (LNPs) containing ionizable amino lipids (iLNPs) are exceptionally potent at delivering siRNA to hepatocytes for gene-silencing applications. • Using in vivo models iLNPs have been optimized for maximum siRNA delivery to hepatocytes through ApoEdependent endocytic pathways.

Clinical trials • The current generation of siRNA iLNPs undergoing clinical trials are well tolerated and show excellent activity against liver protein targets.

Future perspective • Application of the iLNP platform to deliver other nucleic acid therapeutics such as mRNA is under investigation.

References Papers of special note have been highlighted as: • of interest; •• of considerable interest.


Overview of lipid-based drug-delivery systems in the clinic from two leaders in the field.

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Enhancing siRNA delivery by employing lipid nanoparticles.

For several decades extensive research has been conducted into the development of fusogenic lipid nanoparticles (LNPs) capable of introducing large, c...
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