Going beyond the liver: Progress and challenges of targeted delivery of siRNA therapeutics

    Going beyond the liver: Progress and challenges of targeted delivery of siRNA therapeutics Cornelia Lorenzer, Mehrdad Dirin, Anna-Maria Winkler, Volker Baumann, Johannes Winkler PII: DOI: Reference:

S0168-3659(15)00093-0 doi: 10.1016/j.jconrel.2015.02.003 COREL 7552

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

16 December 2014 29 January 2015 2 February 2015

Please cite this article as: Cornelia Lorenzer, Mehrdad Dirin, Anna-Maria Winkler, Volker Baumann, Johannes Winkler, Going beyond the liver: Progress and challenges of targeted delivery of siRNA therapeutics, Journal of Controlled Release (2015), doi: 10.1016/j.jconrel.2015.02.003

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ACCEPTED MANUSCRIPT Going beyond the liver: Progress and challenges of targeted delivery of siRNA therapeutics

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Cornelia Lorenzer1, Mehrdad Dirin1, Anna-Maria Winkler, Volker Baumann, and Johannes Winkler*

These authors contributed equally to this article.

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University of Vienna, Department of Pharmaceutical Chemistry, Althanstraße 14, 1090 Vienna, Austria *Correspondence should be addressed to Johannes Winkler, tel. +431427755058, email [email protected]

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Abstract

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Therapeutic gene silencing promises significant progress in pharmacotherapy, including considerable expansion of the druggable target space and the possibility for treating orphan diseases. Technological hurdles have complicated the efficient use of therapeutic oligonucleotides, and siRNA agents suffer particularly from insufficient pharmacokinetic properties and poor cellular uptake. Intense development and evolution of delivery systems have resulted in preclinically active uptake predominantly in liver tissue, in which practically all nanoparticulate and liposomal delivery systems show the highest accumulation. The most efficacious strategies include liposomes and bioconjugations with N-acetylgalactosamine. Both are in early clinical evaluation stages for treatment of liver-associated diseases.

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Approaches for achieving knockdown in other tissues and tumors have been proven to be more complicated. Selective targeting to tumors may be enabled through careful modulation of physical properties, such as particle size, or by taking advantage of specific targeting ligands. Significant barriers stand between sufficient accumulation in other organs, including endothelial barriers, cellular membranes, and the endosome. The brain, which is shielded by the blood-brain barrier, is of particular interest to facilitate efficient oligonucleotide therapy of neurological diseases. Transcytosis of the blood-brain barrier through receptor-specific docking is investigated to increase accumulation in the central nervous system. In this review, the current clinical status of siRNA therapeutics is summarized, as well as innovative and promising preclinical concepts employing tissue- and tumor-targeted ligands. The requirements and the respective advantages and drawbacks of bioconjugates and ligand-decorated lipid or polymeric particles are discussed. Keywords RNA Interference, siRNA, delivery systems, liposomes, bioconjugates, targeting

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ACCEPTED MANUSCRIPT Introduction – Oligonucleotide Therapeutics

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The concept of RNA interference remains to be hugely promising for therapeutic purposes [1-5]. Taking advantage of the available human genomic data, therapeutic oligonucleotide agents can be designed based on mRNA transcripts sequences. Using careful sequence selection, every single gene can be down regulated by the powerful RNAi mechanism, and even separate transcripts, mutations or splice variants can often be specifically targeted. This new possibility offers quick and easy drug development based on gene functionality, contrasting the complicated search for active agents by traditional means.

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Depending on their mechanism of action, therapeutic oligonucleotides are categorized into several classes: Antisense [6, 7], aptamers [8], splice correcting oligonucleotide [9, 10], siRNA [1], miRNA mimics and antagonists [11], immunomodulating agents [12] and decoys [13] are the most important ones. Because of the incredibly efficient intracellular RNAi machinery, siRNA and miRNA oligonucleotides currently hold the highest promise for therapeutic purposes. Despite their partially contrasting biological effects, the most important features in terms of drug development are shared by all of these compound classes. Oligonucleotides are rather large molecules (at least compared to “classical” small molecule drugs), however much smaller than most proteins such as monoclonal antibodies. Together with their polyanionic and hydrophilic character, the in vivo distribution and cellular uptake are critically and predominantly negatively determined by these traits. In particular, chemical structures and pharmaceutical formulations essentially influence the pharmacodynamic and pharmacokinetic characteristics [7].

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By acting on transcriptional pathways, gene silencing oligonucleotide agents offer new modalities for currently untreatable diseases, including, but not being limited to cancer, viral infections, autoimmune diseases, and cardiovascular disorders. Although these promising properties have been known for decades, there are currently only two oligonucleotide drugs on the market, with one more approved, but no longer marketed. The first drug of this class to reach marketing approval was fomivirsen, an antisense oligonucleotide for treatment of cytomegalovirus (CMV) infections through local application by intraocular injections [14]. Because of low demand caused by the introduction of effective HIV therapies, fomivirsen is no longer available. Pegaptanib is an aptamer available for local therapy of age-related macular degeneration, and antagonizes the detrimental effects of the vascular endothelial growth factor (VEGF) [15]. The antisense oligonucleotide mipomersen has been approved by the FDA in 2013 for therapy of familial hypercholesterolemia. Mipomersen inhibits the transcription of apolipoprotein B 100 and reduces blood levels of cholesterol, LDL-C, and apolipoprotein B [16]. It represents the first systemically applied oligonucleotide. Mipomersen acts mainly in the hepatocytes, and because of its chemical modifications, it shows significant accumulation in the liver. Renal elimination is prevented by extensive binding to plasma proteins. The tendency of mipomersen to induce an elevation of the plasma levels of liver alanine transaminase (ALT) and aspartate transaminase (AST), and hepatosteatosis have triggered controversy during the approval processes on both sides of the atlantic [5]. As the end result, mipomersen was approved in the USA for treatment of severe homozygous familiar cholesterolemia, but only with specific restrictions. In Europe, where the marketing request also encompassed less severe heterozygous forms of familial cholesterolemia, mipomersen failed to achieve approval by the EMEA because of the concerns of toxicity over long application periods [5].

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Mipomersen as a second generation antisense agent is comprised of phosphorothioate and 2’-methoxyethyl modifications. The phosphorothioate backbone is by far the most abundant chemical modification of the antisense class in which one non-bridging oxygen of the phosphate is substituted by a sulfur atom [17]. This little change of the chemical structure influences many pharmacological properties, and accounts for increased stability against enzymatic degradation, and a high proportion of plasma protein binding. On the flip side, the high non-specific protein affinity results in significant off-target effects, meaning biological outcomes that are caused by interactions other than that on the targeted gene [18, 19]. Biological investigations and the clinical evaluation of mipomersen and other antisense agents have recently proven that these off-target effects are indeed translated to considerable relevant side effects [20, 21]. In particular, phosphorothioates result in toxic events at sites of high drug concentrations, such as local reactions at the injection site, and most importantly, hepatotoxicity [22]. Although it is now well known that most of the controversial issues of the antisense class are caused by the phosphorothioate modification, fully appropriate alternatives are lacking. Modifications on the ribose (2’-methyl, methoxyethyl and other alkyl chains, as well as locked nucleic acids (LNA)) [23, 24] aptly increase enzymatic stability, but are only efficacious in combination with at least a partial phosphorothioate backbone, which is necessary for protein binding and thus a certain amount of unassisted cellular uptake [24, 25]. Consequently, it can be expected that next generations of antisense compounds, currently in clinical evaluation, will show similar toxicity, but to a lower extent because of a decrease in dosage afforded by the higher stability.

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Splice-switching oligonucleotides have demonstrated encouraging effects in rare diseases which are characterized by a loss-of-function because of mutations or erroneous gene splicing. The orphan disease Duchenne muscular dystrophy is caused by expression of a nonfunctional dystrophin protein due to deletion of a section of its corresponding gene [26]. As a result, a frame-shift occurs and a non-functional protein is expressed. By blocking a splice junction with an oligonucleotide, the reading frame is restored, and a shortened, but functional protein results. Two oligonucleotides have been developed in parallel, one phosphorothioate, drisapersen [10], and one phosphorodiamidate morpholino oligomer (PMO), eteplirsen [9]. While the latter has shown good clinical outcomes with little or no adverse effects in phase II trials, initially published results have shown a lack of significant clinical benefit for drisapersen in a phase III trial. Follow-up analyses and further extended evaluations are ongoing. To date, no short interfering RNA (siRNA) has yet reached market authorization. This is not surprising, considering the fact that RNA interference was only discovered in 1998 [27], and it took nearly a decade to fully elucidate its mechanism and determine guidelines for sequence design, length, chemical modifications, and formulations [28]. Thus, clinical evaluation of potential drugs has only begun in recent years, with several more promising approaches set to enter clinical trials during the next few years. siRNA therapeutics – efficient reduction of liver targets Just as prior efforts with therapeutic antisense and aptamer oligonucleotides, initial siRNA developments focused on local administration to the eye. An anti-VEGF siRNA, bevasiranib, was evaluated in a phase III trial after application of the unformulated drug [29]. However, poor clinical responses soon led to termination of this trial, emphasizing the need for 3

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thorough and careful preclinical development instead of rushing a poorly designed agent to clinical trials. The main issues yet to be resolved are substrate stability, cellular uptake and endosomal escape, and pharmacokinetics [30, 31]. Being an exogenous nucleic acid, unmodified siRNA is prone to nearly instant enzymatic degradation in body fluids. Variations of the chemical structure at the ribose or the phosphate backbone can significantly increase the stability, and proper modification patterns result in negligible cleavage by nucleases. Although many modifications are useful for increasing stability, only few of those are also apt for keeping intact the pharmocodynamic effect modulated by the RNAi machinery. For therapeutic applications, the most useful siRNA modifications are a limited number of terminal phosphorothioate linkages, and several 2’-O-methyl and/or 2’-F-nucleotides scattered throughout the sequence [32, 33]. These structural optimizations allow the use of unpackaged siRNA by increasing enzymatic stability and preventing nuclease cleavage. Rapid renal filtration and elimination are not avoided [33-36]. Ample experience with antisense compounds have proven that modification with a phosphorothioate backbone evades elimination and increases cellular uptake [17]. In contrast to the antisense technology, a high degree of backbone modifications renders siRNA oligonucleotides unable to activate the RNAi machinery. Consequently, chemical modifications aptly stabilize siRNA, but need an additional targeting approach, either through ligand conjugation or by packaging in a delivery system.

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For improvement of the pharmacokinetic properties and cellular uptake, two distinct approaches seem to be most promising: the direct attachment of receptor-targeted ligands to the oligonucleotide for increasing tissue-specific accumulation and uptake [37], and the packaging in lipid particles [38]. For the latter approach, stepwise evolution of lipid structures and particle composition has resulted in improved in vitro and in vivo efficiency over the years [39]. The currently most advanced siRNA in the clinic is Alnylam’s patisiran (ALN-TTR02), an LNP-packaged siRNA targeted at the transthyretin (ttr) gene [3]. It is developed for treatment of transthyretin mediated amyloidosis, an orphan disease in which mutated proteins are misfolded and build up amyloid deposits. Depending on the mutations locus, the disease results in polyneuropathy and/or cardiomyopathy, the latter form culminates in progressive heart failure. Patisiran efficiently knocked down both wild-type and mutated ttr genes and thus reduces TTR protein production in phase II trials. A sustained effect of an 80 % TTR reduction was achieved for several weeks after a single dose and six months after multiple dosing. The multi-center phase III APOLLO trial for evaluation of clinical effects on neurological symptoms, motor and sensory nerve functions and heart function has begun in late 2013. The same company is developing an anti-TTR-siRNA sequence as a bioconjugate for subcutaneous application for treating TTR-amyloid-induced cardiomyopathies. As targeting ligand, N-acetylgalactosamine (GalNAc) is attached to the sense strand of siRNA using a trivalent linker to achieve receptor-mediated endocytosis via the asialoglycoprotein receptor [40-42]. In phase I test in humans, TTR was reduced in a dose-dependent manner with a maximum of up to 94 % reduction after application of 10 mg/kg siRNA conjugate. The trivalent GalNAc linkage, i.e. attachment of three molecules of the targeting carbohydrate to one siRNA molecule, ensures high affinity to the receptor, decreases the Kd and thus enhances cellular uptake in mouse hepatocytes around ten-fold compared to a divalent conjugate [42]. Biodistribution data in vivo suggest that a high proportion (>50 %) of the applied dose is delivered to the liver. When using bioconjugation to afford siRNA targeting and uptake, the metabolic stability needs to be increased because of the lack of shielding in 4

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extracellular tissues and blood circulation. Thus, chemical modification of the siRNA is more important than for LNP-mediated delivery. A well-attuned combination of nucleotide modifications (2’-O-Me, 2’-F, 2’-desoxy) together with several terminal phosphorothioate linkages not only increases the half-life of the agents, but also enhances the target knockdown efficiency in mice [42]. This was attributed to the higher intracellular (cytosolic) stability resulting in more efficient RNA interference. The encouraging data prompted Alnylam to develop several other siRNA agents as GalNAc conjugates including ALN-AT3 for hemophilia and rare bleeding disorders, ALN-HBV for hepatitis B virus infections, and ALNPCSsc for hypercholesterolemia.

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The carbohydrate GalNAc is also utilized as a targeting agent by Arrowhead Research Corporation for achieving targeting to and uptake into hepatocytes of their anti-HBV siRNA [4]. Instead of covalently attaching GalNAc to the siRNA agent, the ligand is tethered to a carrier molecule, a mellitin-like polymer that is degraded in the acidic environment of endosomes. This so called dynamic polyconjugate carry additional PEG-masking ligands that are likewise cleaved at acidic pH-values, and release the previously masked cationic charge of the polymer. This sophisticated design is supposed to increase endosomal escape of the siRNA through the proton sponge effect, i.e. induction of influx of hydrogen ions followed by water and swelling of the endosomes, finally causing their disruption [43]. While initially presented data on polyconjugates relied on a covalent, but labile attachment of the siRNA cargo to the polymer via a disulfide linkage, more recent evolution of the systems uses simple co-application of siRNA and the carrier [44]. Because little interaction of the uncharged carrier with siRNA can be expected, it is not fully clear to what extent the polymer influences direct siRNA uptake or is simply an adjuvant for increasing endosomal escape. In a transgenic mouse model, efficient reduction to less than 1 % of serum viral markers was detected [4]. Evaluation in humans in a phase II trial has lately been initiated. Early data that have been disseminated indicated reduction of viral markers to around 50 % of the baseline after administration of siRNA at a 2 mg/kg dose. The results of higher doses are not yet published.

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The recently expanding knowledge about microRNAs (miRNA) has quickly prompted the development of miRNA-mimicking oligonucleotides for therapeutic applications [45]. A respective antagonizing phosphorothioate/LNA-modified single stranded oligonucleotide, miravirsen, blocks the interaction of human miR-122 with HCV RNA, an essential step in viral replication [46]. An additional binding to precursor pre- and pri-miR-122 structures and consequent inhibition of nuclease-mediated miRNA processing seem to contribute to the pharmacological effect [47]. Miravirsen is currently undergoing phase II evaluation against HCV infections. It is no surprise that all these clinically evaluated siRNA and miRNA drugs, as well as many antisense agents, are tested for the antagonization of liver targets [48]. The most viable approaches for tissue enrichment and cellular uptake make use of passive (lipid particles) or active (asialoglycoprotein receptor ligand) hepatocyte targeting. Lipid particles are directed to the liver mainly because of their size, and GalNAc conjugates target the respective receptor mainly expressed on hepatocytes. These drugs will be instrumental to prove the principal efficacy of the siRNA technology and gather information about required dosing, target validation, achieved level of gene expression reduction and corresponding clinical effects. Furthermore, comprehensive drug safety assessments will have implications for the whole class of siRNA therapeutics [49, 50]. For making full therapeutic use of the potency of 5

ACCEPTED MANUSCRIPT the technology, it is essential to develop modalities for expansion of the target space to other organs, tissues, and tumors.

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Because of their mode of action, siRNA oligonucleotide agents instantly broaden the druggable target space to all gene transcripts. Nearly all traditional, small molecule drugs rely on distinct binding pockets of proteins. Consequently, the number of potentially druggable targets is limited. The number of disease-relevant targets for small molecules is estimated at around 1000, with less than 30 % of that number currently being exploited for therapy [51]. The advent of therapeutic antibodies has extended the druggable target space, but only to extracellular molecules. Because of interfering with translation, siRNAs can target any desired gene product. Because of the challenges associated with the pharmacokinetics and generally higher costs for development and manufacturing compared to small molecules, the main potential virtue is the antagonization of targets that are not available for small molecules. However, siRNAs currently only work in one direction, that is inhibition of gene translation. As a consequence, development programs preferably focus on diseases unavailable for other treatment options, and on targets with validated overexpression and disease-relevance.

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Despite their intrinsic specificity on the molecular level, gene-silencing oligonucleotides are by no means magic bullet in the sense that side effects are excluded a priori. Both sequencespecific and sequence-unspecific off-target effects are well-documented [19, 52-54]. While the first, caused by hybridization to nucleic acid targets other than the intended, can be avoided by careful oligonucleotide design, the latter is induced by binding to proteins, particularly immune-activating receptors such as toll-like receptors [53]. Side effects can also arise from excessive silencing of the target, especially in non-disease relevant organs and tissues. For example, when attacking a cancer target, it is assumed that the exaggerated expression in tumors is responsible for a higher impact of therapeutic silencing than that in healthy cells. However, since a certain basal expression exists in somatic tissue, the interference with gene expression in unaffected tissue is a cause for concern. Consequently, targeted delivery of siRNA is an important aspect for adding a second layer of specificity, and like for any other therapeutic agent, promises better efficacy with lower adverse effects. Of course, many issues and formulations of targeted siRNA delivery are shared with delivery of other drugs, but there are some specific aspects for oligonucleotide transport: The polyanionic nucleic acid cargo needs to be efficiently packaged and shielded from degradation. After successful cellular uptake, the oligonucleotides must be released into the cytosol in order to exert their therapeutic effect. This is in contrast to most low-molecular weight compounds, for which successful cytosolic transport is often not decisive.

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Application route

Formulation/Modification

Target

Indication

Bevasiranib

intravitreal

-

VEGF

age-related macular degeneration

Opko Health Inc.

Phase III, terminated

[29]

AGN-745 (Sirna027)

intravitreal

-

VEGF

age-related macular degeneration

Allergan/Sirna

Phase II, terminated

[55]

ALN-RSV01

inhalation

-

RSV nucleocapsid gene

RSV infection after lung transplantation

Alnylam Pharmaceuticals

Phase II, completed

[56]

RXI109

intradermal

assymmetric siRNA with phosphorothioates and lipophilic ligands

Connective tissue growth factor

dermal scarring after surgery

RXi Pharmaceuticals

Phase II

[57]

QPI-1002

intravenous

modified siRNA (alternating 2‘-O-Me)

delayed graft function and acute kidney injury

Quark Pharmaceuticals/ Novartis

Phase II

[58]

CALAA-01

intravenous

RONDELTM (cyclodextrinM2 subunit of based formulation with PEG ribonucleotide Solid tumors and transferrin) reductase

Arrowhead Research Corporation

Phase I, completed

[59]

Patisiran (ALN-TTR02)

intravenous

SNALP (stable nucleic acid lipid particles)

transthyretin (TTR)

TTR mediated amyloidosis (FAP)

Alnylam Pharmaceuticals

Phase III

[3]

ALN-TTRsc

subcutaneous

GalNAc conjugate

transthyretin (TTR)

TTR mediated amyloidosis (FAC)

Alnylam Pharmaceuticals

Phase II

[42]

ARC-520

intravenous

dynamic polyconjugate (coinjection with siRNA)

coagulation factor 7 (F7)

hepatitis B

Arrowhead Research Corporation

Phase II

[44]

siRNA-EphA2DOPC

intravenous

liposome (DOPC)

EPHA2

advanced cancers

MD Anderson Cancer Center

Phase I

[60]

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Compound

p53

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Company

Clinical Status

Reference

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modified siRNA (‘Accell’: 2’O-Me, cholesterol, phosphorothioates)

keratin 6a (K6a)

pachyonychia congenita

Atu027

intravenous

AtuPLEX® (liposome) with AtuRNAi® (2'-O-Me)

protein kinase N3 (PKN3)

Atu111

intravenous

DACC lipoplex

Angiopoietin2 (Ang-2)

PF-655

intravitreal

AtuRNAi® (2'-O-Me)

RTP801

Phase I, completed

[61]

advanced solid cancer

Silence Therapeutics

Phase II

[62]

lung indications

Silence Therapeutics

Phase I

[63]

diabetic macular edema (DME)/ AMD

Quark Pharmaceuticals/ Pfizer

Phase II

[64]

Caspase 2

optic nerve atrophy and non-arteritic ischemic optic neuropathy (NAION)

Quark Pharmaceuticals

Phase I/IIa

[65]

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TransDerm

IP

TD101

intradermal injection/ microneedle

intravitreal

modified siRNA (alternating 2‘-O-Me)

siG12D LODER

intratumoral

LODER™ (PLGA matrix)

mutant K-Ras G12D

pancreatic ductal adenocarcinoma

Silenseed

Phase I/II

[66]

TKM-PLK1

hepatic intraarterial/ intravenous

SNALP

PLK1

liver cancer

Tekmira Pharmaceuticals

Phase I/II

[67]

ND-L02-s0201

intravenous

vitamin A coupled lipid nanoparticles

HSP47

fibrosis

Nitto Denko Corporation

Phase I

[68]

DCR-MYC

intravenous

lipid nanoparticles (EnCore)

MYC

hepatocellular carcinoma

Dicerna Pharmaceuticals

Phase I

[69]

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QPI-1007

Table 1. Selection of ongoing clinical trials of siRNA agents. Respective delivery systems and chemical modifications as well as administration routes are listed for illustration of early development (focusing on local administration of unformulated siRNA), and ongoing trials (systemic applications, primarily for liver-associated diseases). 8

ACCEPTED MANUSCRIPT Barriers and biodistribution

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In order to reach their site of action, the cytosol, siRNA therapeutics need to overcome a number of barriers that stand in their way [70]. After systemic (intravenous) application, siRNA agents enter blood circulation and undergo organ distribution, thereby avoiding degradation by nucleases and elimination through the kidney. In organs, the compounds need to extravasate from the blood vessels to the interstitium, overcoming the endothelial barrier [71]. Travelling through the interstitial space, siRNAs are associating to target cells and are taken up generally by endocytotic mechanisms. The endocytic vessels fuse with endosomes, and for successfully attaining the siRNA machinery, the oligonucleotides still have to pass through this last barrier [72, 73]. All of these steps are significantly influenced by the selected delivery vehicle, which has to confer adequate solutions to these diverging challenges.

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Besides intravenous injections, application routes to the blood stream include subcutaneous injection, delivery through the skin, lung [74], or nasal mucosa [75]. Each of those ones add an additional absorption barrier to overcome, so it is no surprise that current clinical attempts focus on intravenous and subcutaneous (for which complete absorption can be expected) injections. First, efficient delivery systems for these routes need to be developed, which can eventually been expanded to other application routes afterwards.

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Its large accessible area and easy administration modes make the skin an attractive target for siRNA application. The outer skin barrier is mainly comprised of the continuous stratum corneum, an impermeable layer of keratinocytes. The most promising possibility for large hydrophilic agents to overcome this barrier is physical disruption by the use of microneedles. Therapeutic siRNAs can be coated onto those stainless steel needles, or injected with microneedle devices [76, 77]. Reports show a good epidermal uptake and gene silencing effect, but little to no absorption into dermal capillaries and systemic circulation. Because the use of needles on diseased skin is associated with pain, their clinical use so far has been limited.

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Similarly, application to the lung restricts siRNA accumulation and action to local effects. Despite a large number of studies reporting the successful use of siRNA in preclinical models, no clinical translation has yet emerged [78]. For intranasal, intratracheal or inhalation delivery systems, lipids (neutral and cationic), polymers (polyethyleneimine (PEI), poly(lacticco-glycolic acid) (PLGA) etc.), and inorganic carriers (calcium phosphate, mesoporous silica particles) have been employed [74]. In the blood and other biological fluids, wild-type nucleic acids are quickly degraded, mainly by exonucleases, which cleave the phosphate linkages between at terminal sites. Endonucleases, cleaving nucleic acids at internal backbone linkages, are less frequent. Consequently, exogenous oligonucleotides are degraded in a stepwise manner starting at terminal sites, yielding shortened oligonucleotides as main metabolites. Both the parent compound and the metabolites are eliminated by renal filtration. The filtration limit for macromolecules lies around 2-4 nm [79], translating to ca. 20-40 kDa for nucleic acids [7]. Standard single and double stranded oligonucleotide (and their metabolites) fail to reach this limit, but increasing the molecular size by either attachment of ligands such as polyethylene glycol (PEG), incorporation into larger particles or significant binding to plasma proteins efficiently save siRNA from elimination [7].

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ACCEPTED MANUSCRIPT On the other end of the size scale, large particles are intercepted by the reticuloendothelial system (RES). Generally, particles above around 200 nm are rapidly cleared by phagocytic cells mainly found in the liver and spleen [80]. However, the RES can be avoided by surface functionalization, such as with amphiphilic PEG chains.

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The endothelial barrier determines distribution into organs [81]. Macromolecules and nanoparticles extravasate primarily by convection, with its relative contribution increasing with transcapillary volume flow. The extent of crossing the endothelial barrier is dependent on the capillary pore size, which generally forms the upper particle size limit [82]. Depending on the organ or tissue, the endothelial gap junctions are differing from very tight (blood brain-barrier) to continuously small (standard arteries and capillaries) and fenestrated (digestive mucosa). The liver, as well as lymphoid tissue and hematopoietic organs, comprises sinusoidal (discontinuous) capillaries. Unlike fenestrations, these capillaries lack a diaphragm over the pore and thus show even superior permeability, allowing exchange of macromolecules. As mentioned before, siRNA delivery systems have an inherent preference to the liver. This pertains to lipid-based particles, cationic and neutral nanoparticles, and lipophilic ligands in bioconjugates. Liver accumulation is caused by the discontinuous nature of the hepatic vasculature (for nanoparticles and protein bound oligonucleotides) or the lipid metabolism (liposomes and lipid or cholesterol conjugates). Within the liver, the use of a specific receptor-targeted ligand can be useful to distinguish between the different hepatic cells and prevent an accumulation in Kupffer cells, the resident macrophages in this organ. Thus, trivalent GalNAc conjugates increase uptake into hepatocytes and consequently gene silencing in this cell type [42]. The asialoglycoprotein receptor is characterized by efficient endocytosis.

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Several diseases show altered capillary permeability in affected organs, and particularly tumors are characterized by increased pore size, thus allowing the passage of larger molecules and particles. This peculiarity is known as enhanced permeability and retention (EPR) effect [83]. Because of the leaky vasculature together with the lack of lymphatic drainage, the EPR effect offers an attractive option for passive targeting of nanoparticles to tumors. Indeed, the EPR effect has been exploited comprehensively by researchers and has been used to explain nanoparticle tumor accumulation in mouse models [84]. On the other hand, passive nanoparticle targeting only amounts to up to 20 % accumulation in tumors (often it is much less), and the extent of vasculature leakage is varying depending on tumor type and other factors [85]. Perivascular tumor growth, intratumoral pressure, and angiogenesis rates all contribute to the heterogenicity of the EPR effect, which varies substantially between tumor model, individual patients and even within a single tumor [86]. Because tumors in animal xenograft models grow much faster than those in humans, the vasculature pores are generally larger in the preclinical models, which often results in an overinterpretation of the EPR effect. A clear clinical proof of its relevance for nanoparticle tumor targeting in a therapeutic setting remains to be shown [83]. The blood-brain barrier (BBB) is characterized by its unique tightness, preventing free diffusion into the brain. For therapeutic purposes, only highly lipophilic molecules with a molecular weight under 400 Da or those with an active transport system can access the brain [87]. Biomacromolecules and untargeted nanoparticle delivery systems suffer from negligible diffusion or transcytosis through the blood-brain barrier. Specific active transport mechanisms for nucleic acids are lacking, and thus other strategies for BBB transcytosis need to be developed. While the BBB exhibits some specific extracellular markers, most if not all endothelial receptors are also found in certain abundance in other endothelial tissues. 10

ACCEPTED MANUSCRIPT To avoid hepatic accumulation and achieve significant siRNA gene silencing in other organs, the use of receptor-specific targeting ligands seem to be mandatory. Cellular uptake and intracellular trafficking

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After extravasation and crossing the interstitium, the cell membrane barrier poses the next hurdle. A lot of data has been generated and discussed regarding this last and maybe most difficult hurdle [88, 89]. It seems clear that the endocytosis pathway is dominating siRNA uptake, whether in naked or conjugated form, or in a delivery system. There are several pathways of endocytosis, the clathrin-coated pit pathway, the caveolar-dependent uptake, clathrin- and caveolin-independent pathways, and macropinocytosis [90-93]. Phagocytosis is restricted to specialized cells, such as macrophages, granulocytes and Kupffer cells. All these uptake mechanisms have been reviewed in depth elsewhere [89, 94]. Although there are small, but important differences between these mechanisms, in nearly all cases of functional uptake, the siRNA agent ends up in an endosomal vesicle [89].

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The crucial point of achieving functionally active siRNA uptake is considered to be endosomal escape rather than endocytosis. Generally, only few molecules of siRNA seem to be transported into the cytosol from early or late endosomes and thus avoid being degraded in lysosomes. There is a reasonably detailed, yet not fully complete understanding of the intracellular endosome trafficking [91]. The endosome system is characterized by a great extent of recycling, i.e. returning to the cell surface. Cargoes taken up through multiple pathways all end up in early endosomes. Early endosomes act as a central sorting place, and define the subsequent fate of the internalized components, either towards recycling back to the surface or maturing into late endosomes and lysosomes and eventually deciding delivery to a target compartment. Association of cytosolic proteins to their side of endosomal membranes is instrumental in their further fate. The Rab family, especially Rab5, is a key regulatory component for this process and, together with other coating proteins, decisive for selection of the targeted cell compartment [95]. Motor proteins are responsible for directed movement along actin- and tubulin filaments to reach the target organelles [96]. Despite the dissection of this fascinating and well-orchestrated intracellular machinery, the gathered knowledge has not yet been translated into smart design of delivery systems that can enhance endosomal escape by rational means. It is largely unclear how and to which extent siRNA delivered in liposomes or polymers or tethered to receptor-specific ligands is eventually reaching the cytosol. Rational attempts to increase endosomal escape have not really relied on the molecular basis of these processes; instead they have tried to make use of physical mechanisms. The so called proton sponge effect is characterized by an influx of hydrogen ions into lysosomes caused by acidification of a basic macromolecule (peptide, polymer, or small molecule) [97, 98]. The subsequent swelling and disruption of the vesicles is intended to result in cytosolic release of its cargo. The induction of membrane fusion events, for example by proteins and peptides which alter their confirmation in the acidic pH of endo- and lysosomes and then fuse with the membranes to increase permeability, is a further strategy to increase cytosolic deliver of siRNA [99]. However, these approaches have not really resulted in a convincing increase of siRNA effects in a therapeutic setting, and safety precautions need to be met. An exploitation of the increasing knowledge of the molecular mechanisms of endosomal sorting and trafficking could certainly help improving the endosomal escape of oligonucleotides in the future. 11

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It is obvious that an interplay of several parameters are decisive for the biodistribution and successful intracellular delivery of siRNA agents, whether as a molecule or in a nanoparticle. These parameters include molecule/particle size, charge, stability eventual surface functionalization, and the presence of a receptor-mediated targeting ligand.

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Based on disease areas with unmet medical need, functional siRNA delivery into tumors [100], the brain [101], and endothelium [99] is especially attractive. For selective targeting, surface receptors with exclusive or highly predominant expression in those organs, tissues, or tumors are required. Because of similarities between endothelial tissues and the heterogeneity of tumor expression patterns, this is by no means a trivial task (Table 2).

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For siRNA delivery across the BBB, the transferrin receptor is most frequently targeted because of its ability to trigger transcytosis [102, 103]. Other targets that have been used for BBB-targeting of siRNA include the insulin receptor [104], and the LDL receptor [105]. Transferrin receptor targeted delivery has shown some success for protein and drug uptake into the brain [102, 106] but in other cases, trapping in the brain microvasculature has been reported [107, 108]. Recently, a more detailed insight into the mechanisms has revealed that the affinity (or avidity) of the peptide or protein ligand to the transferrin receptor is crucial in deciding the fate [103, 106, 109]. High affinity proteins show less brain accumulation than their counterparts with lower affinity to the transferrin receptor. Using in vivo imaging, it was shown that high affinity ligands induced trafficking to lysosomes and degradation [103]. Thereby the level of expressed transferrin receptor was reduced, which even lowered the efficiency of a second dose. A ligand with lower affinity was transported into the brain to a higher extent, obviously by avoiding lysosomal degradation [109].

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A number of delivery systems for nucleic acids have shown to significantly increase brain accumulation several-fold [110]. A selection of peptide and protein targeting ligands (holotransferrin, a transferrin antibody, mutated diphtheria toxin, angiopep-2, and an apoEmimicking peptide) was grafted onto liposomes for blood-brain barrier targeting. Only the liposomes with transferrin antibody resulted in significantly higher brain accumulation, but the amount of brain uptake was still agonizingly low with less than 0.1 % of the injected dose reaching the central nervous system in an in vivo model, and the largest proportion ending up in the liver [110]. No significant decrease of the particles compared to the untargeted system in other organs was detected. It is unclear whether the other four failed due to inefficient binding to the antigen, or other factors such as altered extravasation or insufficient antigen expression in vivo. For gene delivery, a liposome decorated with monoclonal antibodies against the transferrin and insulin receptors and PEG chains for “stealth” delivery were employed in several reports [104]. In a xenograft glioma mouse model, a liposome with two different monoclonal antibodies, one against the rat transferrin receptor, and one against the human insulin receptor, actively transported an expression plasmid for RNA interference to the tumor [111]. Efficient silencing of the targeted EGF-receptor and increased survival time was reported. It is not clear if this approach would be successful for oligonucleotide delivery for which a higher intracellular target accumulation would be necessary. In a recent report, a fusion peptide consisting of a transferrin-targeted sequence and myristoylated transportan was used for condensation and selective brain-uptake of siRNA 12

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[112]. Uptake and silencing in a luminescence assay was increased compared to a peptide lacking the transferrin sequence. Transport across a bEND3 monolayer in a transwell assay was likewise enhanced by the transferrin-targeted peptide. The carrier was not yet evaluated in vivo, and because a certain degree of uptake is possibly mediated by the transportan fraction, the specificity of the system cannot yet be reliably assessed.

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The rabies virus glycoprotein (RVG) was recently used repeatedly to afford siRNA delivery across the blood-brain barrier [113-117]. This 29 amino acid peptide is the required component for attachment and uptake of rabies viruses to host cells [118]. The surface receptor that is responsible for interaction with RVG and the exact mechanism of internalization remain elusive [119]. Several molecules have been identified as potential receptors, including the nicotinic acetyl choline receptor, the neuronal cell adhesion molecule (NCAM), and the low-affinity nerve growth factor receptor (p75NTR). None of those seems to be solely essential for rabies virus uptake and infection, as respective knockout models still show susceptibility to infections [118]. Instead, available data indicates that more than one receptor may be used for neuronal uptake of RVG. It is unclear how RVG mediated transport to the brain, since the identified receptors are localized in neurons, but not at the blood-brain barrier endothelium. Considering the transport of rabies viruses by retrograde axonal transport, a possible explanation could be the uptake into peripheral neurons reaching beyond the blood-brain barrier.

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For utilization of the efficient CNS-targeting properties of RVG, the peptide was fused with a nona-arginine for complexation of nucleic acid cargo [113]. The system specifically transfected neuronal cells, and successfully delivered the siRNA cargo to the brain, where it triggered gene knockdown. Similar systems were generated by attaching the RVG peptides to PEI [115], liposomes [116], and polyamidoamine (PAMAM) dendrimers [117]. When attached to highly charged, spherical PAMAM dendrimers, RVG greatly increased brain accumulation compared to untargeted PAMAM [117]. After tail vein injections, liposomes with RVG grafted to their surface showed high brain accumulation, while liposomes without a targeting ligand or with cRGD were not able to traverse the blood-brain barrier [116]. Likewise, a branched PEI carrier was conjugated with the RVG peptide through disulfide linkages [115]. This systems specifically increased uptake into Neuro2a cell in vitro and significant brain accumulation in vivo. Likely caused by its larger size, considerable uptake into phagocytic liver cells was also observed. Neuronal uptake was blocked by competition with bungarotoxin, which binds to the nicotinic acetylcholine receptor [113]. Uptake was also inhibited by GABA, suggesting a role of the GABAB receptor [117]. The attractiveness of the RVG peptide is further illustrated by its use for targeting of exosomes. These extracellular biological particles are a class of membrane vesicles and contribute to the horizontal transfer of small RNAs between cells [120]. Formed from endosomal compartments within a cell, endocytic vesicles can be released extracellularly to form exosomes. In contrast to other membrane vesicles, exosomes are generally smaller, with a diameter of around 100 nm [121]. Using an exosome system with surface expression of RVG-peptide for acetylcholine receptor targeting, successful siRNA-mediated gene silencing in mouse brain was reported [114]. Both GAPDH and BACE1 genes were shown to be successfully knocked down after intravenous application of RVG-exosomes. Exosomes seem to profit from a lower accumulation rate in the liver, although no comprehensive biodistribution data is available. In the aforementioned study with RVG-exosomes and untargeted exosomes, no down regulation of GAPDH was found in the liver, but a biodistribution study using labeled exosomes was not performed. Thus, it remains unclear 13

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whether exosomes are not accumulated in the liver because of their unique characteristics and/or smaller size than most other delivery systems, or they only fail to release their cargo in liver cells in a sufficient quantity. In another study using intranasal administration, no liver accumulation was detected for both exosomes (30-100 nm) and microparticles (0.5 - 1 µm). While the smaller particles were effectively transported into the brain, the microparticles were deposited in the lung, but failed to reach the bloodstream [122]. EGFR-targeted exosomes injected into the tail vein on the other hand showed significant liver accumulation [123]. Targets and ligands for leukocytes and endothelium

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Integrins are the biggest family of cell adhesion molecules [124]. Being heterodimer proteins consisting of an α and a β subunit, they mediate cell-matrix and cell-cell interactions in physiological and pathological contexts. Their wide expression on cellular surfaces and their constitutive efficient internalization make them attractive targets for receptor-mediated drug transport. Among the 24 integrin family members, β2 and β7 integrins are exclusively expressed on leukocytes [125]. The distribution of α8 in mammalian species is unique among integrins with high abundance in vascular and visceral smooth muscle, kidney glomeruli and alveoli. In the alveolar walls of the rat lung, the cells stained with anti-α8 antibody are likely alveolar myofibroblasts, contractile cells located in the alveolar interstitium. Alveolar myofibroblasts are situated along the capillaries and adjacent to the alveolar epithelium and basement membrane. Other integrins are commonly found on tumor vasculature, and can be targeted for oncologic purposes.

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The tissue distribution of an integrin may provide insight into its function. Although some integrin heterodimers are widely expressed by a variety of cell types (e.g. α2β1, α3β1), others have a restricted distribution. The αIIb subunit is expressed only in platelets and functions as a primary mediator of platelet aggregation. Tumor endothelial cells show an increased expression of αVβ3, and the β2 and β7 subunits are almost exclusively found in leukocytes. The expression of most integrins is developmentally regulated. For example, the α4β1 integrin is widely distributed during embryonic development, but is restricted to leukocytes and endothelial cells in normal adult tissues. For leukocyte targeting, integrin antibody-based specific delivery systems were generated. An antibody targeting the integrin LFA-1 (containing the leukocyte-specific β2 subunit) was fused to a protamine fragment for complexation of siRNA [126]. The system was successful in delivering siRNA in vivo into exogenous K562 cells engineered to express the human LFA-1 receptor. No siRNA uptake was found in murine cells. In a similar approach, an antibody binding to the β7 integrin subunit was grafted onto the surface of liposomes via hyaluronan [127] that was complexed to siRNA via protamine. Both integrin targeted delivery systems resulted in specific gene silencing in leukocytes. Biodistribution of labeled lipid particles was examined and increased accumulation in the inflamed gut about 3.5fold. As with untargeted particles, significant localization in liver and spleen was also found, indicating a particle-sized preference to these organs. The increased expression of αVβ3 integrin on tumor endothelial cells was exploited for delivery of siRNA through a cationic lipid system with cyclic RGD grafted onto its surface [128]. In vitro, the integrin presence was essential for successful uptake and gene silencing, and in vivo, the targeted system resulted in siRNA-mediated gene silencing in tumor 14

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endothelial cells, but not in other endothelia. The biodistribution pattern was altered by attachment of the peptide ligand, and increased accumulation was found in spleen, lung and the kidneys, at the expense of liver accumulation. However, while a relative higher percentage of the dose ended up in other organs, more than 50 % were still localized in hepatic tissues.

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RGD peptides were also attached to the surface of PAMAM dendrimers [129, 130]. Due to the strong inherent binding and transfecting potency of dendrimers, which are mediated by charge interactions with cellular membranes, uptake was not increased through ligand attachment. However, deeper penetration into tumor spheroids resulted by binding of RGD to integrin αVβ3 [130], caused by interfering with the interaction of integrin with the extracellular matrix.

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The cyclic RGD peptide has been attached to siRNA for specific targeting of αvβ3 integrinpositive cells [131]. The cellular uptake was increased with RGD valency, but no clear correlation of uptake with the biological effect was found. Only tri- and tetravalent conjugates actively reduced the targeted luciferase reporter gene, and unlike the results for uptake, a saturation of the effect at an siRNA concentration of 50 nM was detected. Targets and ligands for tumors

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For targeting oligonucleotides to tumors, several other frequently overexpressed surface receptors have been targeted. An ideal receptor for tumor targeting would be only expressed on malignant cells with only very limited expression on somatic cells. Moreover, the receptor needs to be accessible, distributed evenly within the tumor, and bound ligands should be internalized effectively and rapidly. Few receptors aptly fulfill all criteria, and the heterogenicity of tumors with their ability for rapid resistance development further complicates successful tumor targeting. For siRNA delivery, frequently targeted receptors include Her2 [132], the epidermal growth factor receptor EGFR [133], and the epithelial cell adhesion molecule EpCAM [134].

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The folate receptor is also expressed frequently on cancer cells, and is an attractive option for specific targeting. Several reports have employed folate receptor targeting for siRNA using different linkages [135, 136]. A nanoparticulate system consisting of dextran and LPEI was used for folate-receptor specific delivery of siRNA [137]. Both siRNA (via a disulfide bond) and folate were first covalently attached to dextran, before condensation with PEI. In vivo, this system successfully reduced GFP expression in a xenograft model, and compared to siRNA/LPEI complexes, altered the biodistribution pattern towards increased accumulation in the tumor. While the amount found in the liver was unchanged, the increased tumor accumulation was correlated with reduced amounts in spleen. Similar PEI based systems were engineered for folate-mediated targeting and evaluated in vitro or in xenograft in vivo models [135, 138, 139]. Hyaluronic acid is a glycosaminoglycan that is found widely distributed through many tissues, including epithelium and neuronal tissue. It is formed in plasma membranes, is a main component of the extracellular matrix and contributes to cell migration, proliferation and tumor growth. This macromolecule is a ligand for CD44, which is overexpressed in many tumors [140]. Conjugation of hyaluronic acid with a polymeric cationic carrier was used as a delivery system for siRNA for specific tumor targeting [141]. In a xenograft mouse model, a 15

ACCEPTED MANUSCRIPT crosslinked version of this carrier system efficiently delivered siRNA to the tumor site and resulted in gene silencing of the targeted fluorescent reporter protein.

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For Her2-mediated targeting, a recent report used particles consisting of hepatitis B virus L protein, surface antigen (HBsAg), a lipid bilayer, and a Her2-selective affibody [142]. The system showed good in vitro selectivity for Her2-positive cells followed by successful gene silencing. In vivo biodistribution data has not yet been reported.

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For tumor targeting, anisamide, a ligand for sigma receptors, has been used [143]. Grafted to the surface of lipid/protamine siRNA nanoparticles, anisamide afforded intracellular uptake of the siRNA cargo [144]. In a xenograft mouse model, a high extent of tumor localization was found for both targeted and untargeted nanoparticles, and the anisamide ligand did not further increase tumor accumulation. Interestingly, these particles showed much higher accumulation in tumor than in the liver. It is possible that the particle size prevents passage through liver fenestrations, but it may also be caused by the particular xenograft model. Although the ligand did not influence the nanoparticle amount delivered to the tumor, only the anisamide-bearing delivery system resulted in successful gene silencing.

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A trivalent anisamide ligand conjugated to a splice-switching oligonucleotide showed enhanced cellular uptake in human prostate carcinoma cells and a four-fold increased biological effect in a reporter assay cell culture model. Anandamide recognizes the cannabinoid receptor which is highly expressed on neuronal and immune cells. A corresponding siRNA conjugate was successfully taken up in these cultured cells and resulted in down regulation of the siRNA-targeted gene, although high concentrations were required [145].

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Similar to small molecule ligands, peptides can be relatively easily used for attachment to oligonucleotides. Targeting the G-protein coupled receptor BB2, expressed in many carcinoma cell lines, a histidine-rich peptide attached to a splice-switching oligonucleotide was successful in inducing splice correction in a reporter model in a human prostate cancer cell line [146]. However, only modest pharmacological effects were found, and thus the use of this system in a therapeutic setting is doubtful. Several antibody-mediated strategies made use of fusion proteins with basic peptides, such as protamine [126, 147-151]. In these cases, a single chain antibody Fab fragment is fused to a highly cationic peptide to afford siRNA loading through charge complexation. Song et al. were the first to demonstrate antigen-dependent, tissue-specific gene silencing by such a system in vivo [147]. Using a Fab fragment with specificity to the HIV envelope protein gp160, efficient and specific siRNA delivery and effects were found in a xenograft mouse model. Consecutively, similar approaches used single chain antibody fragments for an integrin [126], and the hemagluttinin antigen of the avian influenza virus [149]. Although the fusion proteins are single molecules, they generally form particle-like structures due to the charge complexation formed between the cationic peptides and the nucleic acid cargoes. Thus, they show similar properties as other complexed particles [152], and the fusion proteins can suffer from poor solubility caused by the high cationic charge concentrations. Instead of antibody fragments, alternative protein binder scaffolds offer easier production, higher stability, and more facile derivatization [153]. A Designed Ankyrin Repeat Protein fusion protein (DARPin) with a protamine fragment was shown to successfully and specifically induce gene silencing in an antigen-positive cell line [154].

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Recombinant engineering of self-multiplexing proteins yields well-controlled particles, termed proteinticles, with a uniform size distribution [155]. A fusion protein with an oligonucleotide complexing domain, a cell-penetrating peptide, and a targeting peptide was recently developed for tumor-specific delivery of siRNA [156]. Using EGPF- and vimentintargeting peptides, successful cellular uptake and gene silencing was shown in tumor cell lines in vitro. Although the unloaded proteinticles have a homogenous particle size of around 12 nm, they aggregate to larger and much more heterogenous particles upon complexation with siRNA. Caused by the surface localization of complexing peptides, the nucleic acid cargo induces significant multiplexing of the smaller proteinticles. As a consequence, the targeting peptides, are concealed within the aggregates and thus partially unavailable for receptorbinding. The merit of these proteinticles in an in vivo setting remains to be elucidated.

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Antibody-drug conjugates are attractive molecules for biomedicine development [157, 158]. However, for targeted delivery of siRNA, no successful use has yet been published. Genentech has recently reported the generation and development of analytical methodology for an siRNA-antibody conjugate [159], but to date no pharmacological data of such compounds have been disseminated. The complex generation of site-specific conjugations together with uncertainties about cellular uptake and intracellular trafficking of the siRNA cargo are possible explanation for the lack of published research in this area. Other protein scaffolds offer easier derivatization and conjugation possibilities [160]. Oligonucleotides can be site-specifically attached to protein binders such as affibodies [161], anticalins [162], or DARPins [154, 163]. In the future, such conjugates could be exploited for the optimal combination of specific, highly affine and versatile protein-receptor interaction and siRNA mediated gene silencing.

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Target

transferrin-antibody transferrin-targeted fusion peptide

transferrin receptor

rabies virus glycoprotein (RVG)

not identified nAChR, NCAM, and p75NTR are possible targets

integrin-antibody

integrins

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US antibody affibody anisamide Designed Ankyrin Repeat Protein EGFR-targeted peptide bombesin-like histidine-rich peptide EGFR-, vimentin-targeted peptides

CD44

Her-2 receptor

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hyaluronic acid

folate-receptor

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TUMOR TISSUES

Formulation

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LEUKOCYTES

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sigma receptor

epithelial cell adhesion molecule EGF-receptor

      

liposomes [110] peptide/siRNA complexes [112] peptide/siRNA complexes [113] polyethyleneimine complexes [115] liposomes [116] PAMAM nanoparticles [117] targeted exosomes [114]

 protamine-antibody/siRNA complexes [126]  liposomes [127]          

polythyleneimine complexes [135-137] PEG-siRNA conjugate [138] self-assembled nanoparticles [145] hyaluronic acid-graft-poly(dimethylaminoethyl methacrylate) (HPD) conjugate complexes [140] chitosan/quantum-dot nanoparticles [132] bio-nanocapsule/liposome complexes [141] conjugate [142] lipid/protamine nanoparticles [144] nanocomplexes [154] chitosan-nanoparticles [133]

 conjugate [146]

BB2 EGF-receptor

 proteinticle [156]

Table 2. Targeted siRNA delivery systems for brain, leukocytes, and tumors. Receptor-specific ligands are paired with the respective molecular targets and siRNA- formulations (nanoparticles or bioconjugates).

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ACCEPTED MANUSCRIPT Nanoparticle delivery systems

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A wealth of different multi-componential, macromolecular delivery systems has been developed and evaluated as nucleic acid carriers (Figure 1). Many excellent and detailed reviews have been written on this topic [70, 88, 164-170]. The main categories comprise lipid particles, cationic polymers (PEI, chitosan, polyamines etc.), and cationic peptides. Optimization of these systems by modulation of parameters such as their component structures, composition, encapsulation or complexation method and ratio, has increased their efficiency and often reduced the toxicity. In their native form, these delivery systems all rely on passive targeting in vivo. As described earlier, in correlation to the particle size and surface composition, this inevitably results in high accumulation in kidney, liver and spleen and very limited concentrations in other organs and tissues. In some cases, the EPR effect promotes intratumoral accumulation.

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In individual cases, specific untargeted polymers have been reported to result in significant delivery to distinct organs besides the liver. PEI has a long tradition as a carrier system for gene and oligonucleotide delivery [171]. The nucleic acid cargo is complexed by electrostatic interactions resulting in condensed particles. PEI is a heterogeneous polymer that is characterized by length, degree of branching, and the presence of modifications. Generally, increasing molecular weight is associated with superior cellular uptake, but suffers from toxicity [172, 173]. Recently, a lipid-polymer hybrid generated by reaction of epoxidemodified pentadecane to the amine of PEI600 in a molar ratio of 14:1 [174] was evaluated for its pharmacologic effect in different tissues. Particles generated by condensing siRNA with the polymer and PEG2000-C14 were shown to be effective for gene silencing in endothelial cells without affecting hepatocyte gene expression in mouse models. The endothelial gene silencing was unexpectedly efficient, unlike what has been found for similar, PEI-containing particles. The molecular reasons for the preference of endothelium are not known, but it was speculated that it may be caused by a unique profile of interactions with serum proteins [174].

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Despite these individual reports, it seems to be all but essential to add a ligand for targeted delivery in order to circumvent disposal of these carrier systems in the liver. This certainly increases the complexity of the systems, which in itself are generally consisting of a delicate mixture of several macromolecules [175]. Ligands can be attached to an individual component of the delivery system, for example to one of the lipids making up a liposome, before the final formulation is produced. This approach offers the possibility to purify the ligand-carrier bioconjugate, and often facilitates reproducibility. However, the physicochemical parameters during formulation can be altered by inclusion of a new compound, and it is possible that the receptor-binding capability is reduced because it is not displayed properly on the particle surface. The targeting molecule can also be added after constitution of the final oligonucleotide carrier system by chemical ligation to a reactive group on the carrier surface. A number of attachment chemistries are at the disposal, including thiol-maleimide attachments, amideactive ester couplings, and click chemistry alkin-azide cycloadditions [175]. While this strategy provides straightforward formulation, the ligand density can often vary according to available reactive group on the surface and attachment yields. Usually, it is difficult to assess the actual ligand density afterwards, although the ligand density is important for efficient recognition and internalization [161]. In most cases, multiple ligands will be displayed at the outer surface of the delivery particle. As a consequence, the avidity of the particle is high, 19

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and the off-rate low, even for low-affinity receptor ligands. That means that also binding to other targets (for which the ligand has an inherently low affinity) is a possibility. The example of the transferrin receptor shows that high affinity can lead to a higher extent of lysosomal degradation or entrapment in the microvasculature. Indeed, the amount of transferrin was shown to be critical for successful transcytosis and detachment on the brain side of the blood-brain barrier [101].

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Figure 1. Schematic depiction of modalities for targeted siRNA nanoparticles. Oligonucleotides are either complexed to positively charged polymers or lipids, or encapsulatd in neutral liposomes or polymeric nanoparticles. Complexed cargo can additionally be encapsulated in a lipid membrane. Ligands for targeted delivery are grafted on the surface by using a modified component of the lipid membrane or by post-assembly bioconjugation to reactive surface functionalities. Often PEGylation is applied for reduction of unspecific particle interactions.

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It is important to acknowledge that the presence of a targeting ligand can trigger increased retention and cellular uptake through receptor-mediated endocytosis at the targeted cells, but does not specifically influence the initial tissue distribution. Blood-half life and extravasation into tissue are nearly exclusively determined by the particle size and the physicochemical surface properties of the particles. The largest proportion of nanoparticles is generally found in the liver, kidney and spleen, and accumulation in tumor tissues is dependent on the properties (fenestrations) of the adjacent endothelium. Therefore, if targeted nanoparticles never reach the cell population expressing the respective antigen, the intended specific molecular interaction does not happen at all. On the other hand, surface functionalization alters the physicochemical parameters, and adding ligands can thus even result in reduced accumulation in the targeted tissue.

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An explanation for the often unexpectedly low performance of targeted nanoparticles may be found in the absorption of serum biomolecules around the particle surface, referred to as biomolecular corona [176]. A near monolayer forms the initial biocorona, and its composition is dependent on nanoparticle characteristics and abundances of the distinct biomolecules in the environment [177]. It consists of proteins, but also other molecules like lipids. On top of this, a more dynamic second corona layer with constant molecular exchange is believed to be formed. This biomolecular corona then shifts the nanoparticle properties from its intrinsic values to those conferred to by the environment. Absorbed biomolecules such as apolipoproteins can distinctly influence the pharmacokinetic properties, and interrupt adequate binding of grafted targeting ligands. Surface PEGylation generally decreases the absorption of the biocorona, and thus seems to be crucial to retain the intended targeting properties [176]. Bioconjugates for targeted delivery Being single molecules, siRNA conjugates have very different properties than those packaged into nanoparticle delivery systems. To avoid rapid degradation, the siRNA component usually needs to be chemically modified, with 2’-O-methylation and 2’-fluoridation being the most attractive options [54]. Depending on the size of the attached ligand, renal elimination is a 21

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threat. This can be avoided by increasing the molecular size, for example by PEGylation [178180]. In contrast to particle systems, bioconjugates are more readily distributed into organs, since they are not depending on endothelial fenestrations. Several ligands have been used to further increase hepatic accumulation and uptake, including lipids [181], steroids [182], tocopherol [183], and carbohydrates [184]. Considering the success of the GalNAc platform, it is surprising that the concept has not yet been taken further to achieve accumulation and uptake in other organs.

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Small molecule ligands can be conjugated with relative ease to either the 3’ or the 5’-end of the sense or antisense strand of the siRNA via appropriate linkers that are incorporated during automated oligonucleotide synthesis [37]. The attachment chemistry can be selected from a variation of reactions and adjusted to the distinct ligand. For peptide conjugates, the separately synthesized oligonucleotide and peptide building blocks can be tethered, or a step-wise solid-phase synthesis of the two components can be carried out. Because of differing synthesis procedures and requirements for protecting groups, the latter approach is more complicated, especially when the peptide exceeds the length of a few amino acids [185].

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It can be assumed that the siRNA needs to be liberated from the receptor-specific carrier in order to activate the RNAi machinery. As a consequence, labile linkages that are designed to be cleaved in the reductive, acidic environment of the endosome, are regarded as beneficial. But because the exact mechanisms of intracellular processing of such compounds are not fully understood, the necessity for detachment from the targeting ligand is not confirmed and may be dependent on the specific carrier molecule. The example of the trivalent GalNAc conjugates shows that stable linkages do not prevent efficient gene silencing in all cases.

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Often, multivalent linkages show superior cell binding and uptake characteristics, caused by an increase of avidity by the ability for simultaneous binding to several surface receptors. To achieve this, the spatial arrangement and distance between the ligands is of importance.

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Figure 2. Overview of bioconjugation techniques and ligand structures. Attachment points at the oligonucleotides include primarily the 3’- and 5’terminal hydroxyl functions. Popular linkage types are disulfide as a labile linker to be cleaved in the endosome, and maleimide or triazol linkages derived from click chemistry for stable attachment. 23

ACCEPTED MANUSCRIPT Conclusion

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The decade-long research and development of siRNA delivery systems has taught us important lessons and identified some key parameters. The efficacy of nanoparticle structures relies heavily on size, material characteristics and surface properties. Optimal size is important to prevent rapid elimination (for small particles) and clearance by the reticuloendothelial system (for large particles). Depending on the particular material, an optimal size range of about 50-200 nm has been established. Porosity is a key issue for water penetration, payload release and degradation characteristics. Surface PEGylation helps avoiding immune system activation and capture by immune cells, but generally reduces uptake and cargo release. Cationic charges on the other hand are thought to increase intracellular delivery by enhancing cellular association and by disrupting the endosomal system through their pH buffering capacity, but they also increase non-specific interaction with cells, including those of the immune system, and can cause toxicity.

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These rules help in the design of nanocarriers, but do not encompass all aspects. The immunogenicity and toxicity effects are largely unpredictable, and the extent of successful functional delivery is often surprisingly low. More detailed knowledge of exact cellular uptake events and intracellular trafficking and endosomal sorting is required for improving the rational carrier design [186]. When using receptor-mediated uptake, bioconjugates with high-affinity ligands increase specific cell association and accumulation in the targeted organ or tissue. Similar to particle systems, the extent of cytosolic delivery seems to be heavily dependent on endosomal escape and the cellular sorting machinery. Closer insight is essential for optimal selection of a receptor for targeting and its corresponding ligand as well as finding viable strategies for increasing functional uptake at the extent of lysosomal degradation.

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There is also an increasing translational gap. Academic nanotechnology research often develops increasing complex cutting-edge systems in order to factor in the latest insights of uptake mechanism. Hindered by issues of characterization, manufacturing, and toxicity, many of these sophisticated approaches have ultimately little chance of a translation to a clinical setting. On the other hand, simple reduced solutions have won market approval and have shown improved pharmacotherapy with doxorubicine or paclitaxel, but show little promise for smooth adaptation to biomacromolecule delivery. For siRNA delivery, two modalities have been successful in preclinical and early clinical testing, both for liver targeting: Encapsulation in engineered lipid particles designed for nucleic acids (SNALPs), and conjugates of the cargo with N-acetyl galactosamine, an asialoglycoprotein receptor ligand. Starting from these, an evolution to expand siRNA therapy to other sites seems to be more easily achievable for bioconjugates, for which the receptor ligand can be switched with ease. The progression of lipid particles is ostensibly much more complicated, because the liver accumulation is triggered by both lipophilicity and particle size. While the surface characteristics can be modulated by structural engineering (PEGylation, attachment of targeting moieties), their particle size will still direct them to the liver, and tissues with tight endothelial junctions are all but excluded. Significant progress can possibly be achieved by the development of efficient nanoparticle targeting strategies. The increasing understanding of both basic mechanisms of cellular uptake and trafficking and the structural and biophysical requirements for successful siRNA delivery systems gives reasons to anticipate significant progress in this field in the near future. Ultimately, 24

ACCEPTED MANUSCRIPT successful therapeutic translation and subsequent clinical outcomes within the next decade will decide the impact of siRNA therapies in medicine. Acknowledgements

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We thank Sabine Kalchgruber for drawing a representation of macromolecular delivery systems (Figure 1). Research in our laboratory is supported by grants from the FWF: I519B11 and the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115363, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007–2013) and EFPIA companies’ in kind contribution.

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