Delivery materials for siRNA therapeutics.

INSIGHT | REVIEW ARTICLES PUBLISHED ONLINE: 23 OCTOBER 2013 | DOI: 10.1038/NMAT3765

Delivery materials for siRNA therapeutics Rosemary Kanasty1,2, Joseph Robert Dorkin2,3, Arturo Vegas2,4 and Daniel Anderson1,2,4,5,6* RNA interference (RNAi) has broad potential as a therapeutic to reversibly silence any gene. To achieve the clinical potential of RNAi, delivery materials are required to transport short interfering RNA (siRNA) to the site of action in the cells of target tissues. This Review provides an introduction to the biological challenges that siRNA delivery materials aim to overcome, as well as a discussion of the way that the most effective and clinically advanced classes of siRNA delivery systems, including lipid nanoparticles and siRNA conjugates, are designed to surmount these challenges. The systems that we discuss are diverse in their approaches to the delivery problem, and provide valuable insight to guide the design of future siRNA delivery materials.

S

ince the discovery of RNA interference (RNAi) in mammalian cells, there has been great interest in harnessing this pathway for the treatment of disease. RNAi is an endogenous pathway for post-transcriptional silencing of gene expression that is triggered by double-stranded RNA (dsRNA), including endogenous microRNA (miRNA) and synthetic short interfering RNA (siRNA). By activating this pathway, siRNAs can silence the expression of virtually any gene with high efficiency and specificity, including targets traditionally considered to be ‘undruggable’. The therapeutic potential of this method is far-reaching, and siRNA-based therapeutics are under development for the treatment of diseases ranging from viral infections1,2 to hereditary disorders3 and cancers4,5. Large amounts of effort and capital have been invested in bringing siRNA therapeutics to the market. At least 22 RNAi-based drugs have entered clinical trials (Table 1), and many more are in the developmental pipeline. A key challenge to realizing the broad potential of siRNA-based therapeutics is the need for safe and effective delivery methods. Unmodified siRNA is unstable in the bloodstream, can be immunogenic and does not readily cross membranes to enter cells6. Therefore, chemical modifications and/or delivery materials are required to bring siRNA to its site of action without adverse effects. A broad diversity of materials is under exploration to address the challenges of in vivo delivery, including polymers7, lipids8,9, peptides10, antibodies11, aptamers12,13 and small molecules14,15. Successful systems have been developed by rational design or discovered by using high-throughput screens8,9. Here, we review a selection of promising systems for systemic siRNA delivery, with a focus on the design of the materials and on the approach to the delivery problem. These systems, all with reported in vivo efficacy, are diverse in size, shape, structure, chemistry and mechanism of action. This diversity reflects our still-developing understanding of the mechanisms that underlie much of the delivery process, and highlights the still vast space for innovation and creativity in the field of siRNA delivery.

Delivery challenges

The RNAi pathway is initiated by the presence of dsRNA16. Doublestranded RNA is processed by the enzyme Dicer into 22-nucleotide (nt) pieces with 2-nt overhangs on the 3ʹ ends (Fig. 1). These fragments are loaded into the RNA-induced silencing complex (RISC), the RNA strands are separated, and the passenger strand is degraded. The guide strand remains in the RISC, and the activated RISC–guide-strand complex identifies and cleaves messenger

RNA (mRNA) that is complementary to the guide strand, preventing translation and selectively silencing gene expression (Fig. 1). Synthetic siRNA mimics the structure of Dicer products and is incorporated into the pathway downstream of this enzyme. Although longer dsRNA can also be delivered therapeutically17, siRNA is the most common structure used in RNAi-based therapeutic formulations and is the focus of this Review. To activate the RNAi pathway, siRNA molecules must be delivered to the interior of target cells and be incorporated into the RNAi machinery. As siRNA molecules are too large and too hydrophilic to diffuse across cell membranes alone, delivery material or chemical modification is required to assist their uptake by target cells6. When administered systemically, siRNA faces many additional physiological barriers to reaching its site of action, and delivery systems must be engineered to provide (i) stability against serum nucleases, (ii) evasion of the immune system, (iii) avoidance of non-specific interactions with serum proteins and non-target cells, (iv) prevention of renal clearance, (v) exit from blood vessels to reach target tissues, (vi) cell entry and (vii) incorporation into the RNAi machinery6,18–20. On administration into the bloodstream, naked RNA can be degraded by serum nucleases and can stimulate the innate immune system21–23. A common strategy to address both of these challenges is the chemical modification of the siRNA backbone, often by careful incorporation of 2ʹ-O-methyl or 2ʹ-fluoro modifications, locked or unlocked nucleic acids, or phosphorothioate linkages24 (Fig. 2). Careful design of the siRNA sequence and structure can also help siRNAs to avoid recognition by the innate immune system25. Some delivery systems aim to protect siRNA from degradation and immune recognition by encapsulating it inside nanoparticles26. Whereas nuclease stability and immunogenicity are biological barriers often addressed by modifying the chemical structure of the siRNA itself, additional delivery materials are necessary to surmount other barriers in the body. Interaction with serum components can affect siRNA delivery in various ways. Delivery particles with high positive surface charges can exhibit unfavourable aggregation with erythrocytes27, whereas other delivery systems make use of their interaction with particular proteins to aid uptake by target cells28,29. For example, many liposomal delivery systems, as well as siRNA conjugated to lipophilic molecules, interact with serum lipoproteins and subsequently gain entry into hepatocytes that take up those lipoproteins28. In other cases, serum opsonin proteins can become adsorbed on the surface of delivery vehicles, tagging them for uptake by the mononuclear

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA, 2David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA, 3Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA, 4Department of Anesthesiology, Children’s Hospital Boston, Boston, Massachusetts 02115, USA, 5Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts 02139, USA, 6Institute of Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA. *e-mail: [email protected] 1

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© 2013 Macmillan Publishers Limited. All rights reserved

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REVIEW ARTICLES | INSIGHT

NATURE MATERIALS DOI: 10.1038/NMAT3765

Table 1 | RNAi-based drugs in clinical trials. Drug

Target

Delivery system

Disease

Phase Status

Company

ClinicalTrials.gov identifier

ALN–VSP02

KSP and VEGF

LNP

Solid tumours

I

Completed

Alnylam Pharmaceuticals

NCT01158079

siRNA–EphA2– DOPC

EphA2

LNP

Advanced cancers

I

Recruiting

MD Anderson Cancer Center

NCT01591356

Atu027

PKN3

LNP

Solid tumours

I

Completed

Silence Therapeutics NCT00938574

TKM–080301

PLK1

LNP

Cancer

I

Recruiting

Tekmira Pharmaceutical

NCT01262235

TKM–100201

VP24, VP35, Zaire Ebola L-polymerase

LNP

Ebola-virus infection

I

Recruiting


NCT01518881

ALN–RSV01

RSV nucleocapsid

Naked siRNA

Respiratory syncytial virus infections

II

Completed


NCT00658086

PRO-040201

ApoB

LNP

Hypercholesterolaemia

I

Terminated


NCT00927459

ALN–PCS02

PCSK9

LNP

Hypercholesterolaemia

I

Completed


NCT01437059

ALN–TTR02

TTR

LNP

Transthyretin-mediated amyloidosis

II

Recruiting


NCT01617967

CALAA-01

RRM2

Cyclodextrin NP

Solid tumours

I

Active

Calando Pharmaceuticals

NCT00689065

TD101

K6a (N171K mutation)

Naked siRNA

Pachyonychia congenita

I

Completed

Pachyonychia Congenita Project

NCT00716014

AGN211745

VEGFR1

Naked siRNA

Age-related macular degeneration, choroidal neovascularization

II

Terminated

Allergan

NCT00395057

QPI-1007

CASP2

Naked siRNA

Optic atrophy, non-arteritic anterior ischaemic optic neuropathy

I

Completed

Quark Pharmaceuticals

NCT01064505

I5NP

p53

Naked siRNA

Kidney injury, acute renal failure

I

Completed


NCT00554359

Delayed graft function, complications of kidney transplant

I, II

Recruiting


NCT00802347

PF-655 RTP801 (PF-04523655) (Proprietary target)

Naked siRNA

Choroidal II neovascularization, diabetic retinopathy, diabetic macular oedema

Active


NCT01445899

siG12D LODER

KRAS

LODER polymer

Pancreatic cancer

II

Recruiting

Silenseed

NCT01676259

Bevasiranib

VEGF

Naked siRNA

Diabetic macular oedema, macular degeneration

II

Completed

Opko Health

NCT00306904

SYL1001

TRPV1

Naked siRNA

Ocular pain, dry-eye syndrome

I, II

Recruiting

Sylentis

NCT01776658

SYL040012

ADRB2

Naked siRNA

Ocular hypertension, open-angle glaucoma

II

Recruiting

Sylentis

NCT01739244

CEQ508

CTNNB1

Escherichia colicarrying shRNA

Familial adenomatous polyposis

I, II

Recruiting

Marina Biotech

Unknown

RXi-109

CTGF

Self-delivering RNAi compound

Cicatrix scar prevention

I

Recruiting

RXi Pharmaceuticals NCT01780077

ALN–TTRsc

TTR

siRNA–GalNAc conjugate

Transthyretin-mediated amyloidosis

I

Recruiting


ARC-520

Conserved regions of HBV DPC

HBV

I

Recruiting

Arrowhead Research NCT01872065

NCT01814839

DPC, dynamic polyconjugate; LNP, lipid nanoparticle; NP, nanoparticle; shRNA, short hairpin RNA.

phagocyte system (MPS)18,30. Opsonization and subsequent uptake by the MPS is a common pathway by which delivery systems are cleared from the blood and prevented from reaching their targets. 968

A common strategy for minimizing interaction with serum proteins involves shielding the surface of delivery vehicles with hydrophilic polymers, usually polyethylene glycol (PEG)18,31. PEGylation NATURE MATERIALS | VOL 12 | NOVEMBER 2013 | www.nature.com/naturematerials



INSIGHT | REVIEW ARTICLES

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Figure 1 | RNA interference. Long dsRNA introduced into the cytoplasm is processed by the enzyme Dicer into 22-nt pieces with 2-nt single-stranded overhangs on the 3’ ends. The structure of synthetic siRNA mimics that of Dicer products. The siRNA guide strand is loaded into the RNAinduced silencing complex (RISC), and the passenger strand is cleaved by Argonaute-2 (Ago2). The activated RISC–guide-strand complex identifies and cleaves mRNA that is complementary to the guide strand, preventing translation and thereby silencing gene expression.

of delivery vehicles is aimed at increasing circulation time by minimizing non-specific interactions of particles with serum proteins, cells of the innate immune system and other non-target tissues. One main pathway by which siRNA leaves the bloodstream is through the kidney, where it risks being eliminated in urine. The kidney glomerulus provides a physical filtration barrier that allows water and small molecules to pass into nascent urine while larger molecules are retained in circulation32. The pore size of the glomerular filtration barrier is roughly 8 nm (refs 33,34), and naked siRNA is observed to pass through this barrier into urine35. Many delivery systems aim to be larger than 20 nm to avoid renal clearance36. Notable exceptions include Dynamic PolyConjugates37 (DPCs; 10 nm) and triantennary N-acetylgalactosamine (GalNAc) conjugates3, which are both highly effective delivery systems. It should also be noted that some nanoparticle delivery systems undergo disassembly in the kidney glomerulus38,39. For example, particles that are formed by electrostatic interactions can be broken up when in contact with the negatively charged proteoglycans of the glomerular basement membrane, allowing for passage of siRNA cargo into urine38,39. Delivery systems that are not degraded, phagocytosed or cleared by the kidney must leave the bloodstream by traversing the endothelium to reach other tissues. This occurs most readily in tissues

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Figure 2 | Common RNA-backbone modifications. Careful placement of chemical modifications within the siRNA backbone can improve nuclease stability, and decrease immunogenicity and off-target silencing, without reducing potency. Common modifications to the ribose ring include fluorine (2’-F), methoxy (2’-OMe), locked nucleic acids (LNA) and unlocked nucleic acids (UNA). Phosphodiester linkages between nucleotides are often substituted with phosphorothioate linkages.

whose endothelia are discontinuous, such as the liver and many solid tumours20. Fenestrations in the liver endothelium allow molecules 100–200 nm in diameter to diffuse out of the bloodstream and gain access to hepatocytes and other liver cells40,41. In tumours, highly permeable endothelia are accompanied by poor lymphatic drainage, leading to greater accumulation of circulating nanoparticles in malignant tissue in what is termed the enhanced permeation and retention (EPR) effect42. Most siRNA delivery systems undergo cellular internalization through endocytosis. Various delivery systems aim to improve the rate of cellular uptake by incorporating targeting ligands that bind specifically to receptors on target cells to induce receptor-mediated endocytosis43. Adsorption of serum proteins on the nanoparticle surface may hinder this ligand–receptor interaction44. Other systems use cell-penetrating peptides that can induce cell uptake through endocytosis or non-endocytic mechanisms45. Endocytosed materials are taken up into membrane-bound endocytic vesicles, which fuse with early endosomes and become increasingly acidic as they mature into late endosomes. Some delivery systems incorporate materials that are designed to respond to this low-pH environment by becoming membrane-disruptive in order to trigger the release of siRNA from endosomes into the cytoplasm8,37,46. Still, for many systems the exact mechanism of endosomal release is poorly understood.



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REVIEW ARTICLES | INSIGHT Me =

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Figure 3 | Cyclodextrin polymer nanoparticles. Composition of the cyclodextrin delivery system developed in ref. 49. MW, molecular weight.

On reaching the cytosol, siRNA must be loaded into the RISC machinery to trigger RNAi. The siRNA strand whose 5ʹ end is least stably hybridized is preferentially loaded into the RISC, and the other strand is degraded (Fig. 1). In conjugate delivery systems, attachment of delivery materials to the 5ʹ end of the guide strand is generally avoided because this end is essential for RISC loading47. Conjugation to the passenger strand is usually preferred, although the 3ʹ end of the guide strand has also been used as a conjugation site48. Backbone modifications and siRNA sequences must be judiciously chosen to ensure proper strand selection by the RISC and to avoid partial hybridization to non-target mRNAs, which can lead to off-target gene silencing20.

Cyclodextrin polymer nanoparticles

Cyclodextrin polymer (CDP)-based nanoparticles entered clinical trials for siRNA delivery less than a decade after their introduction5,49. The cyclodextrin delivery system (Fig. 3) was first introduced for plasmid DNA in 1999, with re-optimization of the system for siRNA delivery years later50–57. This was the first targeted nanoparticle siRNA delivery system to enter clinical trials for cancer49. Cyclodextrin polymers are polycationic oligomers (n ≈ 5) synthesized by a step-growth polymerization between diamine-bearing cyclodextrin monomers and dimethyl suberimidate, yielding oligomers with amidine functional groups50. The strong basicity of these amidine groups mediates efficient condensation of nucleic acids with the CDPs at nitrogen/phosphorous ratios as low as 3. End-capping of the polymer termini with imidazole functional 970

groups can aid endosomal escape58, resulting in improved delivery efficacy of both plasmid DNA and siRNA57,59. Although nanocomplexes composed of only CDP and siRNA were able to mediate efficient delivery in vitro, these complexes required additional formulation components for stabilization and efficacy in vivo53,59–61. Both adamantane–PEG (AD–PEG) and adamantane–PEG–transferrin (AD–PEG–Tf) were incorporated to improve particle properties in vivo57,61,62. Adamantane is a hydrophobic molecule that forms a stable inclusion complex with the cyclic core of the cyclodextrin structure. This non-covalent interaction allowed for surface modification of CDP–siRNA nanoparticles with AD-modified excipients by a chemical interaction that is orthogonal to the ionic forces that assist in siRNA binding. PEG shielding was necessary to prevent protein-induced aggregation in serum, but PEGylation also reduced cellular uptake and silencing efficacy. To recover efficacy, the protein transferrin was conjugated to the free end of AD–PEG as a targeting agent. Inclusion of this AD–PEG–Tf conjugate enabled multivalent binding to the CD71 transferrin receptor, improving efficacy63. The CDP–siRNA delivery system has been evaluated in several therapeutically relevant animal models. In a xenograft model for Ewing’s sarcoma57, CDP nanoparticles were formulated with siRNA targeting the oncogenic EWS–FLI1 fusion gene. These induced gene knockdown and had antiproliferative effects with no measured innate immune responses when administered intravenously57. In a syngeneic subcutaneous mouse tumour model, the targeted CDP delivery system showed potent silencing against the validated NATURE MATERIALS | VOL 12 | NOVEMBER 2013 | www.nature.com/naturematerials



NATURE MATERIALS DOI: 10.1038/NMAT3765 cancer target ribonucleotide reductase subunit 2 (RRM2; ref. 64). The clinical translatability of the delivery system was evaluated in cynomolgus monkeys, which indicated that the nanoparticles can be tolerated up to 27 mg siRNA per kg of body weight, with translated efficacy in the range of 0.6–1.2 mg siRNA per kg (ref. 65). Finally, clinical potential was established when RNAi-specific gene inhibition in human melanoma patients (phase-I clinical trial) was shown by monitoring siRNA-mediated cleavage of RRM2 mRNA (ref. 5). Several factors contributed to the translation of this delivery system: (i) the low toxicity of the cationic polymer; (ii) its facile condensation with nucleic acids; (iii) the steric stabilization by PEGylation of the CDP–siRNA nanoparticles in a stable and non-covalent fashion; and (iv) the inclusion of a ligand to improve in vivo uptake and efficacy. These lessons provide useful guidelines for designing future polycationic delivery vehicles.

Lipid nanoparticles

The activity of liposomal siRNA formulations was first reported66 in non-human primates in 2006. Since then, a number of lipid nanoparticle (LNP) RNAi drugs have entered clinical trials (Table 1), including treatments targeting hypercholesterolaemia, transthyretin-mediated amyloidosis and cancer67,68. Before their use with siRNA, liposomes were studied for decades as delivery vectors for DNA-based drugs because of their ability both to protect entrapped oligonucleotides from nuclease degradation and renal clearance, and to promote cellular uptake and endosomal escape69. A number of different lipid and lipid-like structures and formulation methods have been developed, generating a wide variety of LNPs70,71. In the study of these diverse systems, several features have emerged as particularly effective for siRNA delivery. They include the use of cationic or ionizable lipids, shielding lipids, cholesterol and targeting ligands6,72.

Cationic and ionizable lipids. Many liposomes used for siRNA delivery include a cationic or ionizable lipid. Positively charged lipids serve several functions: to improve the entrapment of the negatively charged siRNA, to increase cellular uptake and to aid endosomal escape. Several studies have determined that cationic lipids, which have a constitutive net positive charge, are less efficacious73 and more toxic74 than ionizable lipids, whose charge is dependent on the pH of the surrounding environment. As a result, recent work has focused on the development of new ionizable lipids. The composition of these lipids is generally divided into three parts: an amine head group, a linker group and hydrophobic tails (Fig. 4a). Many of these lipids have been synthesized in a combinatorial manner9,75,76 by altering these three sections systematically in an attempt to develop structure–function correlations. To minimize toxicity without sacrificing efficacy, the pKa of an ionizable lipid should be low enough for it to remain unprotonated during circulation but high enough for it to become protonated in either the early or late endosome. Protonation is necessary to promote membrane fusion and lipid mixing with the anionic lipids in the endosomal membrane77–79. In a study of the effect of lipid pKa on in vivo gene silencing using 53 ionizable lipids, an efficacious range of pKa was found to occur between 5.4 and 7.6. Within this range, efficacy increased as pKa approached an optimum value of 6.44 (ref. 80). Overall liposomal pKa seems to be more important for efficacy than the pKa of individual lipid components. In fact, when two lipids of pKa values 5.64 and 6.93 were coformulated, the mixed liposomes had an overall pKa of 6.93 and exhibited fourfold greater in vivo silencing than liposomes formulated with either lipid alone80. Although there is strong correlation between pKa and efficacy, particles with comparable pKa values can have disparate levels of efficacy in vivo8,77.

Lipid transition temperature refers to the temperature at which lipid membranes shift from the more stable lamellar phase to the less stable hexagonal phase81 (Fig. 4d). This transition promotes destabilization of the endosomal membrane and release of siRNA from both nanoparticles and endosomes82. Lipids with lower transition temperatures, which more readily shift from lamellar to hexagonal phase83 to promote endosomal release, have small polar head groups and large unsaturated hydrophobic tails (Fig. 4b). Lipids with large polar head groups and fully saturated hydrophobic tails are more likely to adopt the stable lamellar phase. Successful lipid formulations are engineered to remain in the lamellar phase during circulation and to transition to the hexagonal phase within endosomal compartments. The structure of a lipid’s hydrophobic tail affects its pKa, transition temperature and potency. In a study of lipids with identical head groups, linkers and tail lengths, increased unsaturation of hydrophobic tails resulted in decreased pKa, decreased transition temperature and increased in vivo efficacy. The most efficacious of the lipids in this study, DLinDMA (Fig. 4c), was subsequently tested in cynomolgus monkeys where it was shown to be capable of silencing 90% of mRNA in hepatocytes66. Variations on the amine head group and linker portions of lipids also affect in vivo efficacy. For the lipid DLinDMA (Fig. 4c), replacing the ether linker with an ester group reduced efficacy whereas a ketal linker improved efficacy8. The amine head group was modified by altering the length of the carbon chain connecting the amine to the ketal group. As the chain length increased, the pKa of the formulation increased and the transition temperature remained constant. Maximal efficacy was obtained using DLin–KC2–DMA (Fig. 4c), which had a pKa of 6.7 (ref. 8). a

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Figure 4 | Lipid structures and shapes. a, Ionizable lipids are composed of three sections: the amine head group, the linker group and the hydrophobic tails. b, Lipids with a small head group and tails composed of unsaturated hydrocarbons tend to adopt a conical structure, whereas lipids with a large head group and saturated tails tend to adopt a cylindrical structure. c, The structures of siRNA delivery lipids DLinDMA and DLin–KC2–DMA. d, The mixing of cationic (orange) and anionic (blue) lipids promotes the transition from the more stable lamellar phase to the less stable hexagonal phase, thus aiding fusion of the liposomal and endosomal membranes.



971

REVIEW ARTICLES | INSIGHT Although many of the ionizable structures examined are twotailed species with a single amine, it has also been demonstrated that multitailed species with numerous amines can be highly efficacious. Several combinatorial libraries have been generated using multiamine head groups bound to numerous hydrophobic tails of various lengths9,75,76.

Shielding lipids. Lipid-anchored PEG is a common component in liposomes. PEG groups serve many purposes: they reduce particle size84,85, prevent aggregation during storage, increase circulation time and reduce uptake by unintended targets such as red blood cells and macrophages85,86. Shielding lipids can also reduce cellular uptake by target cells and have been shown to reduce efficacy both in vitro and in vivo85. After endocytosis, PEG can sterically and electrostatically block the interaction between the liposome and the endosomal membrane, hindering membrane fusion and preventing endosomal release. One strategy for improving the efficacy of PEGylated nanoparticles involves incorporation of acid-sensitive bonds connecting PEG to the liposome. In a comparison of stable carbamate linkers and oxime linkers designed to degrade at pH 5.5, liposomes with oximelinked PEG were stable at pH 7.4 but showed improved release of siRNA at pH 5.5 in vitro. Oxime-linked PEGylated liposomes also demonstrated improved gene silencing in vivo86. Another method to reduce the negative effects of shielding components involves the use of a pH-sensitive modified PEG that binds to liposomes through ionic interactions. The liposomal core consists of 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) as well as 2-hydroxyethyl methacrylate (HEMA)–lysine-modified cholesterol, and the PEG is covalently modified with HEMA–histidine–methacrylic acid. At neutral pH the PEG copolymer has a net negative charge whereas the liposomal core has a net positive charge. In the endosome, imidazole and methacrylic acid residues become protonated, and the net charge of the PEG becomes positive. This results in the release of PEG from the lipid core, exposing the positively charged liposomal membrane and allowing it to fuse with the endosome87. Cholesterol. Many liposomal formulations include cholesterol, which can associate with lipid bilayers88. Up to 25% cholesterol, an increase in the cholesterol content lowers the transition temperature of liposomal membranes containing conical-shaped lipids, aiding their conversion from lamellar to hexagonal phase89. Cationic liposomes with less than 10% cholesterol released less than 5% of entrapped drug within 2 hours when mixed with anionic liposomes, but liposomes with 17% cholesterol released more than 90% of entrapped drug within 5 minutes82. Targeting ligands. To improve the biodistribution of liposomes, many formulations use endogenous or exogenous targeting ligands. An endogenous targeting ligand is a molecule, often a serum protein, that binds to the liposome during circulation and directs the particle to the ligand’s natural cellular target. Exogenous targeting ligands are added to liposomal formulations before injection to bind desired surface proteins on target cells. Advances have been made in developing and understanding both forms of delivery, thereby improving control of the biodistribution of liposomes in vivo. The lipoprotein ApoE has been used as an endogenous targeting ligand by DLin–KC2–DMA-based ionizable liposomes. In wild-type mice, the formulation silenced greater than 90% of factor VII serum protein with a dose of 0.2 mg siRNA per kg. In ApoE knockout mice, however, the same formulation achieved less than 20% silencing at the same dose. Silencing efficacy was restored by incubation of liposomes with recombinant ApoE before injection into ApoE knockout mice29. 972

NATURE MATERIALS DOI: 10.1038/NMAT3765 Retinol binding protein (RBP) is also used as an endogenous targeting ligand. This serum protein binds vitamin A and transports it to cells expressing the RBP receptor, including hepatic stellate and pancreatic stellate cells, as well as myofibroblasts90,91. Delivery of RBP-modified nanoparticles to stellate cells has been reported to have efficacy that is five times as high in cirrhotic rats as in normal rats. These liposomes have been reported to reduce collagen production, thereby reducing liver and pancreatic fibrosis and improving survival rates in rats90,91. The use of exogenous ligands has also been examined as a means of targeting distribution and improving efficacy in vivo. Two notable examples include the small molecule GalNAc, which has been studied as a ligand to target delivery to the liver because of its ability to bind to the asialoglycoprotein receptor on the surface of hepatocytes29, and folate, which has been used to target delivery to rapidly dividing cancer cells92–94. Exogenous ligands are generally attached to the distal end of PEG groups anchored to the delivery system29,92–94. The LNP drug ALN–VSP, under development at Alnylam Pharmaceuticals, is an example of a lipid delivery system that achieves success by careful incorporation of each of these components. The particles contain the ionizable lipids DLin–DMA and DPPC, the PEG-lipid MPEG200–C–DMA, cholesterol, and two siRNAs targeting two genes: vascular endothelial growth factor and kinesin spindle protein95,96. The resulting particles are 80–100 nm in diameter, have essentially no surface charge at physiological pH, and accumulate in the liver and spleen in preclinical models95. ALN–VSP was recently evaluated in phase-I clinical trials for the treatment of advanced solid tumours with liver involvement. In this dose-escalation study the drug was generally well tolerated up to 1.0 mg per kg and demonstrated antitumour activity, including complete response in one patient with endometrial cancer. The RNAi mechanism of action was confirmed by the detection of mRNA cleavage products in tissue biopsies95.

Conjugate delivery systems

A number of promising systems have been developed by directly conjugating delivery material to the siRNA cargo. This approach leads to well-defined, single-component systems that use only equimolar amounts of delivery material and siRNA. The first conjugate delivery systems to show efficacy in vivo consisted of siRNA conjugated to cholesterol14 and other lipophilic molecules28. Other conjugate delivery systems have been developed by attaching siRNA to polymers, peptides, antibodies, aptamers and small molecules48. We will discuss in detail two of the most clinically advanced conjugate platforms, DPCs and GalNAc conjugates, as well as one newly developed system based on oligonucleotide nanoparticles. The first two systems are among the most-developed conjugate delivery systems, and are both drug candidates. Although they are not well represented in the scientific literature because these conjugates are under development by private companies as proprietary technologies, they provide useful lessons for the design of future conjugate delivery materials. Dynamic PolyConjugates. The development of siRNA–polymer conjugate delivery systems designed to respond to intracellular environments — termed Dynamic PolyConjugates — was first reported in 200737. These conjugates incorporate several components, each intended to play a particular role in the delivery process (Fig. 5). The siRNA cargo is attached to a membrane-disrupting polymer by a hydrolysable disulphide linker; the activity of the polymer is masked by PEG side chains while the system is in circulation; to induce uptake by target cells through receptor-mediated endocytosis, ligands are incorporated; the PEG is designed to be shed in the acidic environment inside the endosome, exposing the membrane-active polymer and triggering endosomal release; the disulphide linkage is cleaved in NATURE MATERIALS | VOL 12 | NOVEMBER 2013 | www.nature.com/naturematerials




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Figure 5 | DPC conjugates. DPC materials are designed to respond to the acidic environment of the endosome and the reducing environment of the cytoplasm. In circulation, the membrane-disrupting PBAVE polymer (black) is shielded by PEG. After cell uptake, the PEG chains are shed as the pH of the endosome lowers, exposing the polymer and causing endosomal release. In the cytoplasm, the disulphide bond linking the siRNA to the polymer is reduced, freeing siRNA to trigger RNAi. GalNAc is included in the formulation as a targeting ligand to aid uptake by hepatocytes.

the reducing environment of the cytosol, releasing the siRNA from the delivery polymer; and the siRNA itself is chemically modified to improve nuclease stability and to reduce off-target effects. The membrane-active polymer poly(butyl amino vinyl ether) (PBAVE; Fig. 5) has amphipathic side chains that include alkyl groups interspersed with amines that are reversibly linked to the PEG-shielding agent and the GalNAc targeting ligands. The alkyl chains are important for membrane activity, as longer alkyl chains (propyl or butyl) were shown to improve the polymer’s ability to lyse liposomes in solution97. PEG and targeting ligands are reversibly linked to the polymer backbone using carboxylated dimethyl maleic acid chemistry (Fig. 5), which allows for the release of the PEG-shielding agent in the acidic environment of the endosome. The DPC system was effective at silencing two different genes in the liver when administered intravenously: apolipoprotein B (ApoB) and peroxisome proliferator-activated receptor alpha (ppara). PolyConjugates were designed to target the liver by incorporation of GalNAc ligands, which bind to the asialoglycoprotein receptor (ASGPR) on hepatocytes. Silencing of ApoB was dose-dependent and produced the expected phenotypic effects, including reduction in serum cholesterol37. The importance of individual components of the PolyConjugate was explored, revealing some structure–function insight. The attached GalNAc ligand was essential for both uptake by hepatocytes and in vivo silencing activity. Replacement of GalNAc with glucose greatly reduced hepatocellular uptake, and replacement

with mannose directed uptake to non-parenchymal liver cells that express mannose receptors. In primary hepatocytes, it was reported that the PEG-shielding moiety must be linked to the polymer through a reversible linkage, as attachment with a non-hydrolysable linkage abolished silencing activity37. Newer generations of DPCs are under development at Arrowhead Research Corporation98. The original PBAVE polymers were synthesized by uncontrolled polymerization, resulting in heterogeneity in size and composition. Newer generation of DPC polymers are synthesized using controlled radical polymerizations, including atom-transfer radical polymerization and reversible addition–fragmentation chain transfer, to produce homogenous polymers that are more amenable to optimization98. Hydrolysable bonds are incorporated into different positions, including the polymer backbone and side chains, a strategy aimed at reducing toxicity. The company also reports the development of longer-circulating DPCs by using more stable bonds between the membrane-active polymer and the PEG-shielding agent98. The longer circulation time is intended to aid the targeting of tissues other than the liver. To this end, the use of other classes of targeting ligands has been explored, including peptides, antibodies, small molecules, glycans, lectins and nucleic acids. The latest generation of DPCs have been reported to induce 99% knockdown of liver genes after a single 0.2 mg per kg dose in non-human primates, with the effect lasting nearly 7 weeks98. A 2012 report on DPCs showed that the masked PBAVE polymer could be co-injected with cholesterol–siRNA to induce gene



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REVIEW ARTICLES | INSIGHT silencing in the liver99. Although the polymer was not covalently attached to the siRNA in this case, both were targeted to the hepatocytes and colocalized in endosomes despite the fact that they did not interact with each other in solution. It was demonstrated that the GalNAc ligand, ASGPR and cholesterol moiety were all essential for gene silencing. Interestingly, low-density lipoproteins and lowdensity lipoprotein receptors were not required for silencing (yet they are required if cholesterol–siRNA is injected alone28). This co-injection strategy is reported to be used by the Arrowhead clinical candidate for the treatment of hepatitis B (HBV), ARC-520 (ref. 100). This drug contains two cholesterol–siRNAs targeting conserved regions of HBV transcripts. The original PBAVE polymer has been replaced with a melittin-like peptide with similar reversibly masked endosomolytic properties101,102. The drug is currently in phase-I clinical trials (Table 1). Triantennary GalNAc–siRNA. A liver-targeted siRNA conjugate composed simply of chemically stabilized siRNA with a trivalent targeting ligand has shown promise in the treatment of several diseases. At Alnylam Pharmaceuticals, the conjugates ALN–TTRsc, ALN–PCS and ALN–AT3 are being studied for the treatment of transthyretin amyloidosis, hypercholesterolemia and haemophilia, respectively. In this system the 3ʹ terminus of the siRNA sense strand is attached to three GalNAc molecules by means of a triantennary spacer (Fig. 6). The structure of this delivery material is designed for highaffinity binding to its target, ASGPR, on hepatocytes. Variations of this triantennary ligand have previously been studied for the purposes of targeting drugs or liposomes to the liver103,104, and certain trends relating geometry to binding affinity and cell uptake are documented. Multivalency of the sugar ligand greatly improves cell uptake105,106, and spacing of the sugar moieties also plays a role. In a study of triantennary galactose ligands, binding affinity increased with spacer length over a range of 4–20 Å (ref. 103). Alnylam’s GalNAc conjugate similarly uses a triantennary GalNAc ligand with 20-Å spacing that binds to the ASGPR with high affinity (Kd = 2.48 nM; ref. 107). A comparison of subcutaneous and intravenous administration of this conjugate revealed both greater accumulation of siRNA in the liver and improved knockdown of the target gene by subcutaneous administration107. The three drug candidates based on this conjugate are therefore administered subcutaneously. ALN–TTRsc, designed to silence transthyretin (TTR) for the treatment of TTR-mediated amyloidosis, is the most clinically advanced of Alnylam’s GalNAc conjugates. In non-human primates, ALN–TTRsc administered subcutaneously reduced circulating TTR protein by 70% after one week of daily dosing at 2.5 mg per kg (ref. 3). This level of TTR expression was maintained by weekly administration of the same dose, and serum TTR gradually returned to pre-dosing levels on cessation of treatment. Circulating TTR mRNA levels decreased concomitantly. No evidence of cytokine induction, complement activation or other adverse effects was

NATURE MATERIALS DOI: 10.1038/NMAT3765 observed at a dose of 300 mg per kg, indicating a wide therapeutic window. The expected therapeutic phenotype, the reduction of TTR deposits in peripheral tissues, was confirmed in mice by histology3. ALN–TTRsc recently entered phase-I clinical trials (Table 1). Two other drugs are under investigation using the same GalNAc targeting ligand to deliver siRNA to hepatocytes. By changing the siRNA sequence, this conjugate has been used to silence the expression of two circulating proteins: PCSK9, which plays a role in hepatocellular uptake of low-density lipoprotein, and antithrombin, which regulates thrombin and plays a role in blood clotting. ALN–PCSsc, a conjugate targeting PCSK9 for the treatment of hypercholesterolaemia, showed dose-dependent silencing of the target gene in humanized mice, with a half-maximum effective concentration (EC50) of 0.3 mg per kg (ref. 3). ALN–AT3 targets antithrombin (AT) for the treatment of haemophilia and rare bleeding disorders. A single dose of 1.0 mg per kg ALN–AT3 reduced serum AT protein levels in non-human primates by 50%, and weekly doses as low as 0.5 mg per kg stably reduced serum AT levels by 75–80%. The expected phenotype, increased in serum thrombin levels, was observed to occur in a dose-dependent manner3. Oligonucleotide nanoparticles. The construction of three-dimensional nanoparticles of defined composition from nucleic acids has generated interest because of their unique ability to produce a population of molecularly identical nanoparticles with strictly defined characteristics108. This approach has been adapted to deliver siRNA molecules36,109. Oligonucleotide nanoparticles (ONPs) were composed of complementary DNA fragments designed to hybridize into predefined three-dimensional structures. A previously described method110 of constructing DNA tetrahedra was adapted by incorporating single-stranded overhangs on each edge36. Short interfering RNAs were modified by extension of the 3ʹ sense strands with DNA overhangs that enabled hybridization to the edges of the tetrahedra (Fig. 7). By using unique overhang sequences, six siRNA strands could be attached to each particle, each in a specified position. The resulting nanoparticles had a hydrodynamic diameter of about 29 nm. Oligonucleotide nanoparticles modified with folate ligands were used to study the minimum number of targeting ligands required for delivery and to probe the optimal arrangement of these ligands. These questions are difficult or impossible to address using many other nanoparticle systems. As the position of each siRNA on the tetrahedron could be controlled, siRNAs with or without targeting ligand were assembled into the particles to achieve the desired number and position of folate ligands. A minimum of three folate ligands was required to achieve significant gene silencing, yet incorporation of more than three ligands did not greatly improve silencing efficiency. Furthermore, the positioning of the three ligands was critical: ONPs with three ligands arranged to maximize local density (all three ligands arranged around one side or one vertex) showed efficient silencing, whereas those with ligands distant from HO

O

O

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–

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Figure 6 | GalNAc–siRNA conjugates. Structure of the triantennary GalNAc–siRNA conjugate used in several drug candidates from Alnylam Pharmaceuticals. 974




NATURE MATERIALS DOI: 10.1038/NMAT3765 Six single-stranded oligonucleotides

One-step self-assembly

Six folate-modified siRNAs with 3’ overhangs

Figure 7 | Self-assembly of oligonucleotide nanoparticles. DNA tetrahedra carrying six siRNAs were synthesized in a single step through hybridization of complementary strands36. Positioning of each siRNA on the surface of the tetrahedron could be controlled by using unique sequences in each of the siRNA 3’ overhangs. This precise synthesis method enabled the study of structure–function relationships , such as the effects of the number and spatial orientation of targeting folate ligands.

one another had lower silencing activity. Interestingly, ligand positioning did not affect the rate of cellular uptake, although it was hypothesized that their arrangement might affect the intracellular trafficking of the nanoparticles36. Folate–ONPs were evaluated for both biodistribution and genesilencing ability in mice with folate-receptor-expressing tumour xenografts. After intravenous injection, particles accumulated in both the tumour and kidneys, a pattern consistent with the expression profile of the high-affinity folate receptor111. At a dose of 2.5 mg per kg, folate–ONPs silenced luciferase expression in the tumour by ~60% without significant immunostimulation. The DNA particle was required for this efficacy, as free folate–siRNA demonstrated no significant silencing in this model. Oligonucleotide nanoparticles are set apart from other nanoparticle systems by the exceptional control over particle structure that is afforded by DNA self-assembly. Particle size, shape and surface chemistry are known to influence many aspects of performance19, but the heterogeneity of many systems precludes the study of specific structure–function relationships. Oligonucleotide nanoparticles provide a platform to gain structure–function information that may benefit other delivery technologies.

Outlook

The delivery systems that have shown efficacy in vivo exhibit great diversity in structure, size, chemistry and overall approach to the delivery problem. The systems discussed here are all highly efficacious, promising drug candidates, yet they are each unique in many aspects of their designs. Some have precisely defined structures whereas others are heterogeneous. Their sizes range from hundreds of nanometres to about the size of a single siRNA. Some are held together firmly by covalent bonds or precise hydrogen bonding; others are associated by hydrophobic or ionic interactions. Nanoparticles composed of synthetic lipids are among the most potent formulations in the developmental pipeline. Conjugate systems enjoy precisely defined molecular structures with minimal amounts of delivery material and apparently broad therapeutic windows, and their efficacy continues to improve. Although a variety of delivery systems has been developed in the laboratory, challenges remain in bringing the full potential of RNAi to the clinic. The most advanced systems are nanoparticles formed

by the mixing and self-assembly of various components, yet this formulation method presents additional challenges in the scale-up of the manufacturing process, such as the need for tightly controlled mixing processes to achieve consistent quality of the drug product112. In fact, microfluidic methods are already in use to improve the quality and reproducibility of LNP formulations84,113. Currently, the most clinically advanced systems deliver siRNA to well-perfused tissues such as the liver, where fenestrated or discontinuous endothelium allows the passage of macromolecules to target tissues. But delivery to less accessible tissues remains a considerable challenge. Safe and effective delivery of siRNA to the many tissues where it has therapeutic potential is likely to require development of a variety of systems, each optimized to address the specific challenges of delivery to a particular tissue. As our mechanistic understanding of the delivery process remains incomplete, we have only some guidelines about the characteristics of optimal delivery materials. Nanoparticulate delivery systems aim for particle sizes of 20–200 nm, large enough to avoid renal filtration but small enough to evade phagocytic clearance. Shielding agents such as PEG have proven valuable in preventing non-specific interactions and avoiding immune recognition in circulation. Chemical modification of siRNA is usually used to improve nuclease stability and reduce immunostimulation. Exploitation of endogenous or exogenous targeting ligands is often beneficial to improve uptake by target cells. Membrane-disrupting materials that are shielded or neutralized in circulation but become active in the endosome have proven efficacious. Yet even these established guidelines are not set in stone. For example, it is assumed that siRNA must escape from endosomes in order to trigger RNAi, yet we observe efficient silencing from delivery vehicles, such as GalNAc conjugates, that incorporate no moiety aimed at achieving this. Such delivery systems reach the RNAi machinery by unknown avenues. Indeed, they may follow undiscovered intracellular trafficking pathways, may escape from endosomes by an unidentified mechanism or may enter cells in an unknown non-endocytic manner. Continued research into diverse delivery platforms will help to elucidate the biological phenomena that are currently unclear, and more guidelines will emerge. Indeed, important pieces of the delivery process remain unexplained, and there is much room for creativity and innovation in the design of siRNA delivery materials.



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REVIEW ARTICLES | INSIGHT Received 11 March 2013; accepted 27 August 2013; published online 23 October 2013

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Acknowledgements

The authors acknowledge the service to the MIT community of the late Sean Collier.

Additional information

Reprints and permissions information is available online at www.nature.com/reprints. Correspondence and requests for materials should be addressed to D.A.

Competing financial interests

D.A. has a research grant with Alnylam Pharmaceuticals.



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