Oligonucleotide conjugates – Candidates for gene silencing therapeutics

European Journal of Pharmaceutics and Biopharmaceutics 107 (2016) 321–340

Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

Review article

Oligonucleotide conjugates – Candidates for gene silencing therapeutics Matt Gooding 1, Meenakshi Malhotra 1, James C. Evans, Raphael Darcy, Caitriona M. O’Driscoll ⇑ Pharmacodelivery Group, School of Pharmacy, University College Cork, Cork, Ireland

a r t i c l e

i n f o

Article history: Received 20 May 2016 Revised 24 July 2016 Accepted in revised form 25 July 2016 Available online 10 August 2016 Keywords: Conjugates Bioconjugates Oligonucleotide Gene silencing

a b s t r a c t The potential therapeutic and diagnostic applications of oligonucleotides (ONs) have attracted great attention in recent years. The capability of ONs to selectively inhibit target genes through antisense and RNA interference mechanisms, without causing un-intended sideeffects has led them to be investigated for various biomedical applications, especially for the treatment of viral diseases and cancer. In recent years, many researchers have focused on enhancing the stability and target specificity of ONs by encapsulating/complexing them with polymers or lipid chains to formulate nanoparticles/nanocom plexes/micelles. Also, chemical modification of nucleic acids has emerged as an alternative to impart stability to ONs against nucleases and other degrading enzymes and proteins found in blood. In addition to chemically modifying the nucleic acids directly, another strategy that has emerged, involves conjugating polymers/peptide/aptamers/antibodies/proteins, preferably to the sense strand (30 end) of siRNAs. Conjugation to the siRNA not only enhances the stability and targeting specificity of the siRNA, but also allows for the development of self-administering siRNA formulations, with a much smaller size than what is usually observed for nanoparticle (200 nm). This review concentrates mainly on approaches and studies involving ON-conjugates for biomedical applications. Ó 2016 Elsevier B.V. All rights reserved.

Contents 1.

2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Mechanisms of gene knockdown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Chemical modifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Non-covalent complexation of oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Covalent conjugation of oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Lipids-ON conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Cell penetrating peptides – ON conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Polymers – ON conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Targeting ligands – ON conjugates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Oligonucleotides (ONs) show great potential for therapeutic use due to their ability to bind complementary endogenous messenger ⇑ Corresponding author at: University College Cork, Cavanagh Pharmacy Building, Cork, Ireland. E-mail address: [email protected] (C.M. O’Driscoll). 1 Equal contribution. http://dx.doi.org/10.1016/j.ejpb.2016.07.024 0939-6411/Ó 2016 Elsevier B.V. All rights reserved.

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RNA (mRNA) leading to silencing of specific genes via several possible mechanisms. There are a number of different types of regulatory ONs, including single stranded antisense RNA (asRNA) of 13– 25 nucleotides in length, double stranded small interfering RNA (siRNA) of 20–25 base pairs in length, small nuclear RNA (snRNA) of approximately 150 nucleotides in length, micro RNA (miRNA) of 22 nucleotides in length with short hairpin loops and mRNA of approximately 1500–2000 nucleotides. All of these types of

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oligonucleotide are found in nature and play complex roles in the regulation of gene expression [1]. There has been much discussion about the possibility of using these regulatory mechanisms to treat a wide range of diseases, but despite many years of research two antisense drugs: Fomivirsen (brand name Vitravene) and Mipomersen (brand name Kynamro) have so far been approved, while others are in ongoing clinical trials [2–4]. The reasons for this are largely due to issues with delivery of RNA, which make it unsuitable for therapeutic administration – it is a large, anionic molecule which has poor bioavailability and is highly susceptible to degradation by endogenous nucleases [5]. The size and charge mean that RNA cannot cross the plasma membrane, which it must do to reach its site of action in the cytoplasm or nucleus [5]. Therefore, much research has focussed on finding a suitable delivery system for RNA molecules, which simultaneously protects from nucleases, targets to the target tissue, increases uptake through the plasma membrane and facilitates intracellular trafficking. 1.1. Mechanisms of gene knockdown Regulatory ON molecules bind to their complementary mRNA or pre-mRNA targets via base pairing, but there are several mechanisms by which gene knockdown can occur, depending on the type of regulator and the site of action. Ribonuclease H (RNase H) is a class of enzymes which degrades the RNA moiety in the RNA/DNA duplexes in mammalian cells, and this pathway may be exploited by non-natural ONs to knock down selective genes [6]. The RNase H pathway is most commonly associated with asRNA, wherein RNase H specifically cleaves the 30 -O-P bond of RNA in the DNA/RNA duplex to produce 30 hydroxyl and 50 phosphate terminated products [7]. The majority of ON drugs currently in clinical trials, including Fomivirsen and Mipomersen make use of the RNase H pathway, and are usually chemically modified to prevent degradation in vivo and have increased bioavailability [1]. Another possible mechanism that involves degradation of the mRNA is the RNA interference (RNAi) pathway, which is activated by siRNA and miRNA. siRNAs are short 21–23 base pair double stranded ONs, which are processed from long dsRNA in the cytoplasm by an endoribonuclease enzyme called Dicer. The processed siRNAs then act catalytically by binding to proteins, which make up the RNA-induced silencing complex (RISC) containing the Argonaute 2 (Ago2) enzyme. The activation of RISC is induced upon unwinding of the sense/antisense strand and thermodynamic selection of antisense strand. The activated RISC with the antisense strand binds to the complementary mRNAs with high sequence similarity, targeting the mRNA for degradation, thereby inhibiting the protein translation [5]. In contrast, miRNA has a hairpin structure and does not bear full complementarity with the target strand, binding only 6–8 nucleotides in the 30 untranslated region of the mRNA [8]. Unlike siRNAs, miRNA are processed from dsRNA in the nucleus by an endoribonuclease enzyme (Drosha) and transported to the cytoplasm by the nuclear exportin-5 miRNA [9]. Once in the cytoplasm miRNA undergoes cleavage by Dicer to form siRNAs, activating the RISC complex, but gene silencing is thought to occur via blocking of translation or by sequestering the mRNA into P-bodies where other degradation enzymes act upon it [8]. Since miRNA is not sequence specific, one sequence may regulate many genes. For this reason, there has been less interest in using miRNA for therapeutic uses. Gene silencing may also be achieved at the mRNA level by nondegrading mechanisms. Splice switching oligonucleotides (SSOs) are short, synthetic, antisense, modified oligonucleotides that bind to splice junctions on pre-mRNA, thereby blocking the RNA-RNA base pairing or RNA-protein binding that occurs between the splic-

ing machinery and pre-mRNA [10]. This results in an alternative splice mRNA product, which in turn leads to a different protein sequence being translated [11]. This type of RNA-regulating ON has the potential to treat diseases caused by incorrect mRNA splicing, and has shown particular promise in the treatment of Duchenne Muscular Dystrophy (DMD) [11].

1.2. Chemical modifications As mentioned previously, a major barrier to the use of ONs as therapeutic agents is the difficulty in delivering them to the site of action [12]. Naked ONs have a short half-life in vivo due to attack by nuclease enzymes, renal excretion, and accumulation in the liver and kidneys [12]. These issues of bio-distribution have partially been addressed by chemical modifications on the ONs, and in addition these modifications are widely used to improve uptake. The backbone of the ON chain is often changed to a phosphorothioate (PS) in which the oxygen anion is replaced by sulfur and increases nuclease resistance as well as decreasing renal clearance due to increased binding to serum proteins [13]. PS-ONs were one of the first class of chemically modified ONs to be developed, but they suffer from low bioavailability and off-target effects [14]. Substitution at the 20 position of the nucleoside sugar by methyl (OMe) or methoxyethyl (MOE) groups increases binding affinity to the target mRNA, as well as decreasing serum protein binding [15], and these modifications are used in several antisense drugs currently in clinical trials [4]. However, these 20 -O substitutions on antisense oligonucleotides inhibit RNase H activity on the target strand, and therefore their use must be limited in order to retain the gene silencing effect [16]. This is often achieved by using gapmers, in which unmodified nucleosides are flanked by regions of 20 -O-substituted bases [16]. These types of chemical modifications are also used in siRNA to improve nuclease resistance, in addition to 50 -O-methyl substitution of the sense strand, which prevents this strand from binding to RISC [17]. Other chemical modifications involve more complex changes to the ON backbone. Locked nucleic acids (LNAs) include a methylene bridge between the 20 -O and the 40 -C of the sugar, which result in much higher binding affinity and enzyme resistance [18]. There are several LNA drug candidates in clinical trials, including Miravirsen which suppresses production of a miRNA (miR-122) involved in the life cycle of the hepatitis C virus [19]. LNAs may also be used in siRNA to confer higher stability and functionality [20]. Peptide nucleic acids (PNAs) consist of a neutral, peptide-like backbone instead of ribose sugars, and similarly phosphorodiamidate morpholino oligomers (PMOs) substitute the ribose sugar for a morpholino ring. These ONs possess high resistance to nucleases and low binding to serum proteins due to their neutral backbones [21]. However, their failure to activate RNase H means that they are used either as gapmers or in functions which do not require mRNA degradation, such as splice switching ONs. Several PMOs are being evaluated at different phases of clinical trials, for example AVI-4126, AVI-4065, AVI-4557. These PMOs have been evaluated pre-clinically and/or as first-in human trial, as therapeutic interventions for the treatment of restenosis, Hepatitis C virus or downregulation of cytochrome P450, respectively [22,23]. The most recent, GRN163L, a phosphothioamidate oligonucleotide conjugated to a lipid was shown to inhibit telomerase, limiting the lifespan of human pancreatic cells [24]. Table 1 lists studies highlighting a variety of conjugate linkages used to conjugate an oligonucleotide (PNA/ASO/PMO) to peptide ligands/polymers/ small molecules for various biological applications, specifically focusing on gene silencing. Another review published recently focuses on nucleic acid bioconjugates, specifically focusing on the cancer therapy and detection [25].

Table 1 List of oligonucleotide (PNA/ASO/PMO) conjugates with peptides/ligands/polymers along with their conjugate strategy. Most of these studies involved chemical modification of oligonucleotides for enhanced stability against nuclease degradation. The use of polymer was favored for prolonged stability/retention and enhanced cellular uptake for biological applications. Polymer/peptide/ligand

Gene target

ODN - chemical modification

Linkage

Conjugate strategy

In vitro

In vivo

Refs.

PMO

N/A

Amidation

Peptide was conjugated on the 50 end of the oligonucleotide

C2C12 mouse myoblasts

Mdx mice

[68,69]

PNA

Murine dystrophin gene Bcl2

177

Amidation

[117]

PNA

miR122a

Amidation

N/A

[118]

TAT peptide

PMO

N/A

Fluorescein modified oligonucleotide Phosphoramidation and Cystamine modification

Human CLL/SLL cell line Mec-1 MDA-MB-231 cells

N/A

Polyarginine

A549 cells

N/A

[119]

DOTA-Tyr3–octreotate peptide Polyarginine

PNA

BCl2

Radiolabeled PNA

Amidation

N/A

miRNA210

N/A

Amidation

K562 cells

Dogs with B-Cell lymphoma N/A

[120,121]

PNA

[122]

CPP

PMO

N/A

Amidation

Various bacterial cells

N/A

[66]

Antenna peptide

PNA

Gyrase A RNA supFG1 gene

Amidation

AV16

AV supFG1 transgenic mice

[123]

Small peptides

PNA

PTEN mRNA

N-terminus of the PNA was modified with FITC and Rhod N/A

Carboxyl group in DOTA-Tyr3-octreotate is conjugated to the amine group on PNA Boc protected PNA was conjugated with the amine of peptide 50 end of nucleic acid was conjugated to the Nterminal of peptide 50 end of nucleic acid was modified with Cystamine and conjugated with the TAT peptide using a linker Carboxyl group in DOTA-Tyr3-octreotate is conjugated to the amine group on PNA Carboxyl terminal of PNA was conjugated with the polyarginine using solid phase synthesis Peptide was covalently linked to the primary amine at the 30 end of PMO The peptide was covalently linked to the PNA at the C-terminal lysine

BCL1 cells

Balb/c mice

[124]

Small peptides

PNA

N/A

Amidation

CHO, HEK and MDCK cells

N/A

[125]

PEO Ruthenocene

PNA PNA

Nociceptin/ orphanin FQ receptor mRNA RpoB N/A

Boc protected PNA was conjugated with the amine of peptide Boc protected PNA was conjugated with the amine of peptide

Amidation Amidation

HATU and PyOxim coupling HATU coupling

[126] [127]

PEG-PNA-PEG

Amidation

[128]

PNA

N/A

Amidation

HEK293 cells

N/A

[129]

Peptide, chromophore, proteins, biotin derivatives Apolipoprotein E peptide variants

PNA

N/A

N/A

Phosphoramidation

PNA was conjugated with PEG on either side following amidation reaction Amine of PNA was conjugated with the carboxyl end of small molecule via solid phase synthesis Phosphoric group at the 50 terminal of PNA was conjugated to the amine of small molecules

N/A

Dinuclear rhenium (Luminescent agent)

hsa-mir1323 N/A

N/A HT29, HeLa, HepG2, and PT45 cell lines N/A

N/A N/A

HGG.Cu and DETA

N/A Acetylated at the N-terminal N/A

N/A

N/A

[130]

PMO

Mouse dystrophin exon-23

Biotinylated Pip6aSH-PMO

Amidation

Biotinylated Pip6a-SH was conjugated to the 30 -end of the PMO through its C-terminal carboxyl group

C2C12 cells

N/A

[131]

N/A

32

Alkyne-Azide addition

Alkyne modified peptides was conjugated to the azide modified oligonucleotides

A549 cells

N/A

[132]

N/A

N/A

N/A

[133,134]

Azido connector peptide on PMO

N-terminal of the peptide was conjugated to 50 end of the PolyT-20 oligonucleotide Peptide modified PMO was conjugated to the alkyne functionalize peptide

N/A

Mouse dystrophin exon-23

Azide-alkyne addition Azide-alkyne addition

Murine H2k mdx myoblasts

N/A

[135]

Amidation linkage M12 and MSP peptides/ Chimeric Arginine rich (RXR)4 – MSP peptide DOTA-Tyr3-octreotate

Azide-alkyne addition (click) TAT peptide 30 primer DNA and TW171–17 RNA Small peptide sequences Poly T-20 and PNAs Apolipoprotein E peptide PMO variants

Lu-labeled

P-labeled at 50 -end

Phosphoramidation and amidation

Amidation

M. Gooding et al. / European Journal of Pharmaceutics and Biopharmaceutics 107 (2016) 321–340

Oligonucleotide

(continued on next page) 323

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Table 1 (continued) Oligonucleotide

Gene target

ODN - chemical modification

Linkage

Conjugate strategy

In vitro

In vivo

Refs.

2,20 -dipicolylamine (DPA-a radiolabel agent)

PNA

N/A

N/A

Azide-alkyne addition

N/A

Wistar rats and nude mice

[136]

CPP

PNA

N/A

Alkyne-azide addition

HeLa pLuc705 cells

N/A

[80]

CPP

PNA

STAT1 gene

C-terminal of the PNA was modified with a biotin tag N/A

DPA is modified with azide functionality and is conjugated to Alkyne modified PNA (solid phase synthesis) N-terminal Azide modified PNA and was coupled with the N-terminal alkyne on the peptide TAMRA-PTD-4-Hal(traziole-Gly-PNA)conjugate was established

Endothelial cells

N/A

[81]

ASO

Luciferase

Phosphorothioate

Disulfide Linkage

A549 cells

N/A

[137]

PNA

Luciferase

N/A

Disulfide linkage

HeLa pLuc705 cells

N/A

[138]

FAM-RGD-RGD-p53 peptide

DNA

N/A

Disulfide linkage

HeLa cells

N/A

[139]

Asialofetuin (AF)

DNA and PNA

[140]

dsDNA

N/A

Thioether Linkage

Murine primary hepatocytes and human HepG2 hepatocarcinoma cells N/A

Male C57BL/6 mice

Small peptide sequence

RNA template (telomerase activity) N/A

50 thiol modified ODN N/A

N/A

[141]

Histidine rich-peptide (H5WYG)

ASO

Luciferase

20 O-methylated

HeLa pLuc705 cells

N/A

[142]

Bombesin (BBN) peptide

ASO

Luciferase

20 -O-Me phosphorothioate

Amino-ethoxyethyl linker and Maleimide linker Thioether linkage

PC3 cells

N/A

[106]

RGD peptide

SSO

N/A

Thioether linkage

A375/eGFP654 cells

N/A

[103]

PEI

PNA

Luciferase

20 O-Me oligonucleotides terminated with a thiol at 50 end N/A

Thiol modified oligonucleotides were conjugated with Maleimide modified BBN peptide Thiol modified ODN is reacted with the Maleimide modified cyclic RGD peptide

HeLa pLuc705 cells

N/A

[93]

Gold nanoparticles

ASO and ONs

EGFP

Tertra-thiol or Mono-thiol modified ONs

Thioether linkage

PEI is modified with a heterobifunctional amine reactive SPDP linkers that conjugates with the Cysteine terminated PNA Thiol-modified or cyclic–disulfide modified ASOs were added to citrate stabilized gold nanoparticles

C166, C166-GFPsii, NIH 3T3, RAW264.7, and HeLa cells

N/A

[143]

Others Triantennary GalNAc (GN3)

ASO

SRB1, ApoCIII and human TTR

Short S-cEt (S-20 -OEt-20 ,40 -bridged nucleic acid) gapmer ASOs

UnyLinkerTM support

GN3 was conjugated to the 30 end of oligonucleotide

Mouse hepatocytes

[144]

PNA

Luciferase

NA

Cholesterol hemisuccinate (solution conjugation) Bisphosphonate (Fmoc strategy) PEI (amine and thiol reactive SPDP PEG8 heterobifunctinal) CPP (solid phase synthesis)

N-terminal of PNA was conjugated with different molecules and with different strategies

HeLa cells

C57BL/6, Human APOC3 transgenic mice and TTRIle84Ser transgenic mice N/A

Disulfide/thioether linkage a-Aminoisobutyric acid (Aib)-containing peptide sequences CPP (8-Arg)

Cholesterol-, bisphosphonate, PEI, CPP

1,3-dipolar Huisgen cycloaddition reaction

Thioether linkage

Thioether linkage

The 3-nitro-2-pyridylthio-activated peptide was reacted with the 50 end of the thiol modified oligonucleotide PNA contains NPys-Cys functionality and the peptide a C-terminal free Cys functionality The sulfhydryl groups on ODN were activated by aldrithiol-2 before the addition of peptide DNA is conjugated to AF using SMCC linker and PNA is hybridized to the DNA-AF assembly

N-terminus of peptide conjugated to 50 thiol terminated oligonucleotide Peptide was conjugated to the 50 -end of Oligonucleotide

[145]

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Polymer/peptide/ligand

M. Gooding et al. / European Journal of Pharmaceutics and Biopharmaceutics 107 (2016) 321–340

325

[82] C. elegans J774A cells Phosphine of small molecule conjugated with the hydroxyls of ONs in solid phase synthesis

[147] N/A PC3 cells

[146] N/A N/A

N-succinimidyl ester of the quinone methide precursor was reacted with pPNA Phosphine of small molecule conjugated with the hydroxyls of ONs in solid phase synthesis

N/A

SSO

PNA

Mono- and tri-valent anisamide molecules

CPP (KFF, ANT, TAT, (RXR)4XB and (RFR)4XB

RNA polymerase a-subunit

2 -O-Me phosphorothioate

N-succinimidyl ester as a linker Phosphorousoxygen bond formation Phosphorousoxygen bond formation 0

N/A PNA Quinone methide

DNA sequence Luciferase

Oligonucleotide Polymer/peptide/ligand

Table 1 (continued)

Gene target

ODN - chemical modification

Linkage

Conjugate strategy

In vitro

In vivo

Refs.

1.3. Non-covalent complexation of oligonucleotides In addition to chemical modification, a widely researched method to overcome many of the barriers to ON delivery is the use of cationic materials to bind to the anionic ONs and shield them from enzymatic degradation, as well as preventing interaction with serum proteins and aiding cellular uptake [26]. One of the simplest cationic materials that have been widely used to coat ONs is polyethyleneimine (PEI), which may consist of primary, secondary and tertiary amines in various proportions depending on the level of branching. This polymer has been used to deliver various ONs with high efficiency due its high ON binding affinity, and the ability of PEI to promote endosomal escape via the ‘proton sponge’ effect [27]. However, PEI alone shows little potential for therapeutic use due to its high toxicity caused by membrane disruption and initiation of apoptosis [28]. Cell penetrating peptides (CPPs) are a class of peptides that have the ability to cross the cell membrane via an endocytic mechanism [29]. These peptides are usually cationic, making them attractive candidates for ON delivery agents as they are able to form electrostatic complexes, which are taken up by cells. Noncovalent complexation with CPPs has been widely investigated for the delivery of siRNA, and early studies made use of peptides such as penetratin, TAT and MPG to deliver siRNA in vitro and in vivo [30–32]. Novel CPPs such as EB1 and Pepfect-6 have also been engineered to enhance endosomal escape in order to increase gene knockdown [31,33]. Furthermore, CPP fusion proteins have been produced which combine the cell-entry attributes with the properties of other peptides to enhance oligonucleotide complexation. For example, TAT has been produced with an attached double stranded RNA-binding domain (DRBD) which increases siRNA binding [34], and octaarginine has been expressed as a fusion protein with the RVG peptide which acts as a targeting ligand for the CNS and promotes uptake across the blood-brain barrier (BBB) [35,36]. Cationic complexation of ONs has also been achieved using cationic lipids which encapsulate ONs into liposomes. This approach has been widely demonstrated using a wide range of lipid formulations, which often include amphoteric lipids such as dioleoyl phosphatidylethanolamine (DOPE) and 1,2-distearoyl-snglycero-3-phosphocholine (DSPC), which assist in destabilizing the endosomal membrane [26]. Several commercially available lipofection reagents have been developed for research purposes, such as Lipofectamine which is regarded as a ‘gold standard’ for in vitro cell transfection studies. However, these reagents are unsuitable for therapeutic applications due to high cell toxicity [37,38]. More recently lipids have been used to form lipid nanoparticles (LNPs) containing siRNA which are highly effective at in vivo delivery [39]. Several of these LNPs are currently in clinical trials including ALN-TTR02 from Alnylam to treat transthyretinmediated amyloidosis, and ALN-PCS to treat high cholesterol by reducing expression of the enzyme PCSK9 (www.alnylam.com). Despite the large amount of interest and research into noncovalent particles for the delivery of ONs, there have so far been no approved drugs using this technology, and clinical trials of nanoparticles have experienced many setbacks, such as the termination of Arrowhead’s CALAA-01 clinical trial, a targeted nanoparticle system containing cyclodextrin for the treatment of melanoma [40]. Non-covalent systems are complicated, often containing several different components, and therefore their manufacture is difficult to replicate on a large scale [41]. There are also problems associated with complex stability and immunogenicity caused by one or more components of the system [41]. An alternative approach is the covalent conjugation of ONs to deliveryenhancing components to generate more well-defined molecules which bear more resemblance to traditional small molecule drugs

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as well as avoid the complications of toxic cationic species. Many moieties have been conjugated to ONs in order to enhance delivery including lipids, CPPs, polymers and targeting ligands [42–44], and the remainder of this review will focus on these conjugates.

2. Covalent conjugation of oligonucleotides 2.1. Lipids-ON conjugates Covalent conjugation of siRNA to cholesterol and other lipophilic molecules has been shown to promote uptake and RNAimediated knockdown of target genes including the apolipoprotein B (apoB) protein [45] and Huntingtin (Htt) [46]. The attachment of lipophiles to siRNA promotes binding to high-and low-density lipoproteins (HDL and LDL) and leads to cell uptake via interaction with lipid receptors [47]. Cholesterol-siRNA conjugates premixed with LDL or HDL displayed higher knockdown of the protein than the un-complexed conjugate. Lipid complexation also affects the bio-distribution of siRNA conjugates, with LDL binding favouring higher uptake in the liver, and HDL binding leading to uptake in other tissues including the kidney, heart, adrenal and lung. Affinity for lipoproteins is related to the length of the alkyl chain of the conjugate lipid, with C22 and C18 lipids showing the strongest binding to HDL and consequently the highest level of gene silencing [47]. siRNA conjugates of a-tocopherol (vitamin E) have also been shown to successfully knock down genes in the liver [48] as well as the brain [49]. In the first study, knockdown was shown to be unsuccessful in the absence of serum, suggesting a similar lipoprotein-dependent mechanism to the cholesterol conjugates. In the brain study, the siRNA conjugates were complexed with HDL prior to delivery, and the authors postulated that neuronal cell entry was via LDL receptor-mediated endocytosis [49]. Another recent study that explored the conjugate of vitamin E and siRNA, delivered siRNA against PLK1, the conjugate formulation was further conjugated with Herceptin as a ligand to target breast cancer cells. The study showed 88% reduction in PLK1 mRNA levels and

when used in combination with docetaxel, the formulation performed 75 fold better in suppressing the cancer growth in comparison with docetaxel alone [50]. The conjugation of an siRNA against vascular endothelial growth factor (VEGF) to estrone showed accumulation in the liver, possibly due to lipoprotein binding, but in addition showed targeting to the mammary glands which was explained by the authors as binding to the estrogen receptor [51]. This suggests that estrone may be used as a targeting ligand, in particular for certain types of cancer in which the estrogen receptor is overexpressed. Most RNA-lipid conjugates have been prepared using a stepwise solid-phase synthesis approach, despite this method being significantly more expensive and complicated than solution synthesis, although it has the advantage of easier purification and fewer solubility concerns [52]. Conjugation is usually directed to the 30 or 50 end of the sense strand, since the antisense strand is incorporated into RISC and therefore to modify this strand would sterically interfere with the formation of the complex [53]. Conjugations at the 30 end are usually achieved using a solid support modified with a bifunctional linker which allows attachment of the required lipid group, followed by extension of the oligonucleotide chain by phosphoramidite chemistry (Fig. 1A). Examples of such linkers are homoserine [54], 4-hydroxyprolinol [45] and glycerol [55]. Conjugation to the 50 end of an ON strand is more straightforward since solid phase synthesis takes place in the 3–50 direction and therefore a lipid can be introduced via a phosphoramidite reagent [56–58]. In addition, the 1,3-dipolar cycloaddition between an azide and an alkyne, the so-called click chemistry, may also be used to conjugate an alkyne-bearing lipid to an azide-containing base (Fig. 1B). The base may then be converted to a phosphoramidite and inserted at the 50 end of an ON [59,60]. 2.2. Cell penetrating peptides – ON conjugates In addition to the formation of non-covalent complexes described earlier, CPPs have also been covalently conjugated to ONs. CPP covalent conjugates of negatively charged ONs such as pDNA and siRNA have been shown to be ineffective since a single

Fig. 1. Examples of lipid conjugation to ONs at the 30 end via a homoserine linker (A) [54] and 50 end via ‘click’ chemistry (B) [60].

M. Gooding et al. / European Journal of Pharmaceutics and Biopharmaceutics 107 (2016) 321–340

conjugated peptide is not sufficient to neutralize the negative charge on the ON to allow interaction with the anionic cell membrane. In addition, anionic ONs may form large, insoluble aggregates with cationic CPPs which are not taken up by cells [44]. Early studies suggested that siRNA conjugates of penetratin and transportan were successful in eliciting significant knockdown in mouse fibroblasts [61] or hippocampal neuronal cells [62]. However, in both of these studies, the conjugates were not rigorously purified, which has raised queries suggesting that the active species may have been the siRNA-peptide electrostatic complexes rather than the conjugates [63]. A later study compared purified siRNA conjugates of the CPPs TAT and penetratin to a siRNAcholesterol conjugate in vivo [64]. It was shown that the peptide conjugates did not knock down the target gene significantly more than siRNA alone and furthermore, the conjugates induced high levels of the immune marker proteins TNFa and IL-12p40 [64]. Therefore, most recent studies have made use of non-covalent electrostatic complexes which have shown more promising results for anionic ONs. Table 2 lists the studies of siRNA covalently conjugated to ligands/peptides. Conversely, for neutral ONs such as PMOs or PNAs, there have been some very successful studies carried out with covalently conjugated CPPs [65,66]. In particular, splice-correcting PMOs that correct the mutation in the dystrophin gene in Duchenne Muscular Dystrophy (DMD) have been enhanced by conjugation to various CPPs. PMOs are conjugated to peptides in solution by functionalization of the 50 end of the PMO with a piperazine ring, followed by coupling to the C-terminus of the peptide using 2-(1H-benzotria zole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBT) to activate the carboxylic acid (Fig. 2A) [67]. Several peptide-PMOs (PPMOs) have been formed using a (RxR)4 peptide, in which 6-aminohexanoic acid linkers (x) were inserted into an octaarginine peptide in order to increase stability in blood circulation [68,69] (Table 1). When conjugated to a splice-correcting ON, this peptide was successful in recovering dystrophin in a DMD mouse model, except in cardiac muscle [70–72]. Subsequently, another peptide, termed the ‘B-peptide’, was developed with the sequence (RxRRBR)2 where one of the x groups is substituted with b-alanine (B). The PPMOs formed with this peptide were shown to be less toxic in an in vitro study [73], and in vivo displayed the ability to restore dystrophin in the heart [74]. B-peptide conjugates were taken into preclinical trials by AVI Biopharma (now Sarepta Therapeutics) as AVI-5038; however, tests in monkeys showed that the PPMOs resulted in significant kidney toxicity after 4 weeks of weekly 9 mg/kg injections [75]. In an attempt to limit kidney localization, a chimera peptide was produced by combining the B-peptide with a muscle-specific heptapeptide (MSP) which displayed enhanced splice correction activity compared to B-peptide alone [76,77]. The muscle-specific PPMO was shown to be non-toxic in mice at a biweekly dose of 6 mg/kg which increased dystrophin to >20% in skeletal muscle and <5% in cardiac muscle [78], although trials in higher mammals are required to confirm an improvement in toxicity over AVI-5036. PNAs have also been used as SSOs as potential DMD therapeutics. PNA internalization peptides (PIPs) were designed by combining elements of (RxR)4 and an arginine-modified version of the CPP penetratin, and the resulting PNA conjugates showed effective cellular uptake in vitro as well as successful exon skipping activity in a DMD mouse model [79]. In this case, the CPPs were synthesized with a C-terminal cysteine to allow conjugation with the PNA via either a disulfide or thioether conjugation in solution (Fig. 2B and Table 1). A recent study has attempted to simplify the often laborious procedure of producing multiple CPP conjugates by outlining a method for synthesizing a library of CPP-PNA compounds using copper-catalyzed Huisgen ‘‘click”

327

chemistry [80,81]. A PNA was synthesized on solid phase with an N-terminal azide and a C-terminal cysteine which is subsequently linked to a biotin group via a disulfide bond (Table 1). A library of different CPPs was subsequently produced with N-terminal alkyne groups which may be easily conjugated to the PNA via a click reaction catalyzed by copper (II) sulfate and sodium ascorbate. The conjugates are easily purified by washing on a streptavidin support (Fig. 2C). The resulting library of conjugates may then be screened in cellular assays in order to identify the most effective peptides to deliver maximum cellular uptake and splice correcting activity [80]. Another recent study reported the antimicrobial effect of PNA when conjugated with 5 different types of CPPs, namely KFF, ANT, TAT, (RXR)4XB and (RFR)4XB via ‘‘O” linker, as a treatment for intracellular infections. The study showed most efficient inhibition of RNA polymerase a-subunit (rpoA) of the intracellular pathogen Listeria monocytogenes with PNA conjugated TAT, (RXR)4XB and (RFR)4XB only [82]. 2.3. Polymers – ON conjugates Attempts to enhance the delivery of ONs have also included the covalent conjugation of various polymers. Attachment of poly (ethylene) glycol (PEG) – termed PEGylation – is well known to improve the bioavailability of drugs, by increasing the resistance to endogenous enzymes as well as reducing immunogenicity [83]. Conjugation of PEG with a variety of different ONs has been achieved including siRNA [84–86], antisense ONs [87] and PNAs [88]. PEGylation of siRNA has been shown to increase circulation time in mice and decrease renal clearance [84] as well as reduce immunogenicity [85]; however, the non-cleavable attachment via ‘click’ triazole or thioester linkages of long PEG chains in the 5–20 kDa range leads to an inhibition of gene silencing proportional to the PEG molecular weight. This inhibition may be overcome by using a cleavable linker such as a disulfide bond which is cleaved in the endosome [89,90]. Alternatively, a recent study has investigated the use of short, monodisperse PEG12 units conjugated to siRNA or antisense ONs via a non-cleavable peptide linkage [87,91]. These modified ONs showed no reduction in activity compared to unmodified controls, although as the authors point out, these short PEG chains are probably insufficient to significantly affect bio-distribution or plasma stability. Table 2 details more recent studies on PEG-siRNA conjugates using a cleavable disulfide linkage strategy. Cationic polymers have also been used in order to enhance cellular uptake of ONs, in a similar way to CPPs. Polyethyleneimine (PEI) is a highly cationic polymer and an effective, although very toxic, transfection agent which promotes disruption of the endosomal membrane via the ‘proton sponge’ effect [92]. The primary amine groups of a branched PEI were modified with a splicecorrecting PNA using a heterobifunctional PEG linker bearing a N-hydroxysuccinimide (NHS) ester to attach to PEI and a pyridylthiol group to form cleavable disulfide bonds with the N-terminal cysteine of the PNA [93]. The modified PNA successfully induced splice correction in an in vitro assay and was up to 10-fold more effective than an analogous PNA-octaarginine conjugate, although significant toxicity was seen even at micromolar doses [93]. Another study conjugated small polyamines to the 20 position of phosphorothioate ONs and showed that longer chain polyamines lead to an increase in intracellular antisense activity by enhancing binding affinity with the target gene through electrostatic interactions [94]. It is also speculated that longer polyamines may also increase cell uptake via ON charge neutralization. A set of more elaborate siRNA-polymer conjugates, termed Dynamic Polyconjugates (DPCs), were first presented in 2007 to deliver siRNA to mouse liver cells [95]. These compounds are based around the amphiphilic polymer poly(butyl amino vinyl ether)

Target ligand

RNAi gene target

Polymer

Type of conjugation

In vitro

In vivo

Comments

Refs.

Amidation PGE(2)-siRNA conjugate

PGE(2)

Fas and GFP

1,6-diaminohexane and cystamine bisacrylamide (poly(DAH/ CBA)

Amidation

H9C2 cells

N/A

 " Cellular uptake with the conjugated PGE2-Fas siRNA polyplex  The conjugate PGE2-Fas siRNA polyplex, reduced the amount of apoptotic cells in hypoxic conditions (24.6 ± 3.9%), when compared with untreated control (63.5 ± 5.7%)

[148]

Azide-alkyne linkage (click) Folate-PEG-siRNA Folate conjugates

EGFP

Polycationic polymer

Azide-alkyne addition

KB/eGFP-luc cells

N/A

 " Cellular uptake (in vitro) with folate targeted conjugates  ; (80%) mRNA level (in vitro) with Folate-PEG-siGFP conjugates complexed with a cationic polymer containing succinoyl-tetraethylenpentamine units connected by lysine

[149]

siRNA-PEG conjugates

N/A

EGFP

Lipofectamine

Azide-alkyne addition

H1299 cells

BALB/c nude mice

   

; (60%) in mRNA level in vitro with PEG5k-siLNA ; (55%) in mRNA level with PEG10k-siLNA ; (50%) in mRNA level with PEG20k-siLNA " (50%) Enhanced siRNA retention (1 h) after i.v injection with PEG20k-siLNA conjugate  ; Bladder accumulation of PEG20k-siLNA

[84]

Small molecule ligandsiRNA conjugates

N/A

Luciferase

N/A

Azide-alkyne addition

HeLa cells

N/A

 It was shown that CuAAC conjugation of azido-functionalized small molecule ligands to solid-support bound oligo-ribonucleotide was the most efficient reaction and may yield an efficient high throughput synthesis method  siRNA conjugates showed 75% KD of luciferase gene

[60]

PAsp(DET)-siRNA conjugate

N/A

Cy3-labeled scrambled siRNA and PLK1 siRNA

Polycation (Polyaspartamide derivative with two repeating units of aminoethylene in each side chain

Azide-alkyne addition

SKOV3-Luc cells A549 cells

N/A

 " Cellular uptake of conjugated siRNA PICs in SK-OV-3 cells compared to mono-siRNA PICs  Conjugated siRNA PICs showed improved endosomal escape than mono siRNA PICs  ; (70%) Cell viability with conjugated siRNA PICs targeted against PLK1 gene in A549 cells

[150]

Peptide-siRNA conjugate

HN-1TYR (tumorspecifically internalizing peptide)

hRRM2

N/A

Azide alkyne addition

NHDF, KB, MCF-7, MDA-MB-468 and ZR-75-1 cells

N/A

 " Cellular uptake of HN-1TYR in ZR-75-1, MCF-7 and MDA-MB-468 cells  ; (50%) RRM2 protein levels with HN-1TYR-anti-hRRM2 siRNA in MCF-7 cells

[151]

Disulfide and thioether linkage HAP-SS-siRNA Hyaluronic acid

GAPDH

Polyethylenimine

Disulfide linkage

BEL-7402

N/A

 " Transfection efficiency with the conjugate MnHAP/ siRNA  ; Cell viability with the conjugate MnHAP/siRNA complexes  Comparable GAPDH KD with the conjugate and Lipofectamine as confirmed by western blot

[152]

PLGA-conjugate-siRNA

STAT3

Chitosan

Disulfide linkage

SKOV3

N/A

 STAT3-siRNA-PLGA/CSO micelles showed knockdown of STAT3 of 0.22 ± 0.13 at gene level and 38.0 ± 1.0% at protein level  " Cellular uptake (91.9 ± 6.1%)  ; Cell viability  " Cell apoptosis

[153]

N/A

M. Gooding et al. / European Journal of Pharmaceutics and Biopharmaceutics 107 (2016) 321–340

Conjugate

328

Table 2 List of siRNA conjugates with targeting ligand or lipid molecules. In most studies the siRNA conjugate was formulated into a nanoparticle using a cationic polymer/lipid for complexation to enhance cellular uptake/endosomal escape and gene silencing effect both in vitro and in vivo.

Table 2 (continued) Target ligand

RNAi gene target

Polymer

Type of conjugation

In vitro

In vivo

Comments

Refs.

siRNA-phospholipids conjugate

N/A

PLK1

PLGA, DSPE-PEG2000 and DDBA (cationic lipids)

Disulfide linkage

HeLa cells

BALB/c nude mice

 ; (64%) PLK1 mRNA level with siPlk1-PCNPs conjugate  " (76.4%) Apoptosis  ; Cell viability with co-delivery of docetaxel and Plk1 siRNA  ; (70%) PLK1 mRNA level in vivo with siPlk1-PCNPs/DOX  ; Tumor volume (6 fold smaller than the control)

[154]

THIOMAB siRNA conjugates

Antibody

PPIB

N/A

Thioether and disulfide linkage

PC3, 293 and Igrov-1

Nude mice

 Co-Knockdown of the endocytic component HSP4 doubled the silencing efficiency of the conjugate, antiTENB2-siPPIB ARCs  IV injection of the conjugate, anti-TENB2-siPPIB ARC in PC3-TENB2-high cells xenograft mouse model resulted in 33% reduction of PPIB mRNA 5 days post injection

[155]

Poly(amide) polymersiRNA conjugate

GalNAc

Sci10-ApoB

N/A

Disulfide linkage

HepG2 cells

Sprague Dawley rats

 " 70–90% ApoB mRNA knockdown efficiency at doses of 1–3 mg/kg in mice liver was exhibited by High molecular weight copolymers (24.6 kDa or 38.6 kDa)  " Clearance from body with Poly (amide) polymer conjugates, i.e. less accumulation of material from repeated dosing

[97,98]

Molecular umbrella siRNA conjugate

N/A

Sjögren syndrome type B antigen (SSB) Luciferase

N/A

Disulfide linkage

Hek293 cells

Norway Rat

 siRNA-umbrella conjugate mediated dose-dependent gene knockdown in the absence of transfection agent lipofectamine  ; (32%) in SSB mRNA levels following intra-vitreal injection into rat retina at 100 lg/eye dose

[156]

PP75-siRNA conjugate

N/A

Stathmin

PP75 - L-phenylalanine grafted onto poly(Llysine isophthalamide)

Disulfide linkage

U251 cells

BALB/c nude mice

 PP75 mediates pH dependent release from the endosomes by pore-formation, following acidification  ; (81%) mRNA level: in vitro and ; (90%) protein levels: in vitro  ; Tumor volume In vivo with Co-delivery of the chemotherapeutic agent carmustine (via i.p.) and PP75-stathmin siRNA conjugate

[157]

PEG2000-PE (Phosphothiol ethanol) conjugated to siRNA

N/A

N/A PEG-PE micelles

Disulfide linkage

N/A

 GFP silencing in C166 endothelial cells showed 28% reduction in target gene expression when treated with siRNA-PE/PEG-PE at a ratio of 1:750 w/w ratio  When survivin siRNA was used in the conjugate, there was a 70%, 50% and 50% reduction in the viabilities of A2780, MB-231 and SKOV 3 cells respectively. In contrast, there was only 20% reduction in viability in paclitaxel resistant SKOV3-tr cells  Survivin siRNA-S-S-PE and paclitaxel co-loaded in polymeric micelles. This led to an approximate 60% reduction in cell viability with 200 nM siRNA and 40 nM paclitaxel

[158,159]

HA-siRNA conjugate

Hyaluronic acid

Linear-poly (ethyleneimine)

Disulfide linkage

Balb/c mice

 ; (60%) gene KD (in vitro) with HA-siRNA/LPEI complexes with cleavable linkers, while only (20–40%) gene KD was observed with complexes containing non-cleavable linkers  In vivo: HA-ApoB siRNA/LPEI complexes showed dose dependent reduction in ApoB mRNA in the liver, with approximately 60% reduction for the highest treatment dose (30 lg) versus no detectable reduction in complexes lacking HA

[160]

- Survivin - EGFP

ApoB

- A2780, MDAMB231 and SKOV3 cells - C166 cells

MB-231 cells

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Conjugate

330

Table 2 (continued) Target ligand

RNAi gene target

Polymer

Type of conjugation

In vitro

In vivo

Comments

Refs.

TPGS (Vitamin E) siRNA conjugates

Herceptin

PLK1

N/A

Disulfide linkage

NIH3T3, MCF7, and SK-BR-3 cells

N/A

 ; (88%) mRNA levels at 125 nM of Plk1 siRNA (formulated in TPGS micelles) in SK-BR-3 cells  When conjugated to the antibody Herceptin the IC50 (lg/mL) of TPGS micelles with DOX + Plk1 siRNA was reduced over 75 fold compared to free docetaxel at 72 h in SK-BR-3 cells

[50]

Antibody-siRNA conjugates

Antibodies CD79b and TMEFF2

PPIB

N/A

Thioether and disulfide linkage

PC3 cells

BALB/c nude mice

 The authors describe a method for using a real time polymerase chain reaction (RT-PCR) assay coupled with an antigen capture step to quantify intact antibody siRNA conjugates

[109]

Polycations-PEG-Folic acid

Folic acid

Luciferase

Polycation, containing 8 succinoyl tetraethylene pentamine (stp) building blocks

Disulfide linkage: siRNA was conjugated to Inf7

KB cells

Rj: NMRInu (nu/nu) mice

 ; (85–90%) in luciferase gene expression (in vitro), when treated with the treatment formulation  In vivo studies indicated a high tolerability and no discernible accumulation in non-targeted tissues

[161]

siRNA-macromer conjugates

N/A

Luciferase

Cationic (PRINT) hydrogel

Disulfide and acrylamide macromers

HeLa-Luc cells

N/A

 " siRNA protection against 10% FBS over 48 h, when covalently incorporated in PRINT hydrogel particles  An amine monomer (AEM) was incorporated to aid in uptake and endosomal escape. 30% AEM-containing PRINT hydrogel particles provided the ideal combination of gene silencing efficiency (EC50 OF 17.5 nM siRNA) and cyto-compatibility

[162]

siRNA albumin conjugation

Albumin

IGF-IR

N/A

Thioether linkage

N/A

BALB/c nude mice and Sprague Dawley rats

 " Pharmacokinetic parameters of the albumin conjugated siRNA  " Greater ability of uptake by capillary endothelial cells and vascular smooth muscle cells  ; (40%) in IGF-1R mRNA levels in the aorta. In contrast, there was a significant reduction of 50% (p < 0.05) IGF-1R mRNA in the kidneys with the unconjugated siRNA (with conjugated showing only a minimal reduction)

[163]

ScFv Antibody conjugated siRNA

Transferrin receptor

Survivin

Poly-L-lysine

Disulfide linkage

U87 cells

Nude mice

 ; In vitro colony formation assay (from 90 to 40)  " Apoptotic levels from <5% to >35%  ; In vivo surviving protein levels (U87 intracranial tumors)  " Median survival time of mice treated with antibody conjugate from 47 days to 54 days

[110]

Albumin-siRNA conjugates

BSA

IGF-IR

N/A

Thioether linkage

N/A

N/A

 A significant batch-to-batch variability was observed while conjugating maleimide-functionalized siRNA to the cysteine residue of BSA  It was discovered that the siRNA desalted using ammonium acetate was a poor choice, as ammonium ions would become counterions to the polyanionic siRNA  Ultrafiltration prior to activation with SMCC significantly increased the yield of siRNA-albumin conjugate by approximately 15-fold

[164]

PLGA-siRNA conjugate

N/A

EGFP

Linear Poly (ethyleneimine)

Disulfide linkage

MDA-MB-435-GFP cells

N/A

 " Cellular uptake of siRNA-PLGA conjugates coated with either LPEI25k or LPEI2.5k compared to siRNA-LPEI complexes alone in MDA-MB-435-GFP cells  ; (42.9 ± 4.6% and 51.6 ± 2.7%) mRNA levels (in vitro) with siRNA—PLGA coated with 25k or 2.5k LPEI, respectively

[165]

M. Gooding et al. / European Journal of Pharmaceutics and Biopharmaceutics 107 (2016) 321–340

Conjugate

Table 2 (continued) Target ligand

RNAi gene target

Polymer

Type of conjugation

In vitro

In vivo

Comments

Refs.

Dual gene targeted (DGT) multi-siRNA conjugates Single gene targeted (SGT) multi-siRNA conjugates

N/A

VEGF, GFP, Survivin, Bcl-2

Linear poly (ethyleneimine)

Thioether and disulfide linkage

MDA-MB-435, PC3, A549, PBMCs, HeLa and MCF-7 cells

N/A

 ; GFP and VEGF expression levels more effective with DGT multi-siRNA conjugates than SGT multi-siRNA conjugates transfected with LPEI 25k  Next Bcl-2 and survivin were crosslinked and used to treat HeLa and MCF-7 cells  ; Cell viability (HeLa) reduced to 46.5 ± 5.6% for DGT multi-siRNA and 76.0 ± 9.2% for the SGT multi-siRNA mixture  " (40%) Apoptotic cells (HeLa) with DGT multi-siRNA, compared with less than 20% for the SGT multi-siRNA mixture. This enhanced apoptotic effect was also seen in the MCF-7 cells

[166]

Di and tri block copolymers developed from PEG-siRNA conjugates

N/A

- EGFP - EGFP and VEGF

Solid lipid nanoparticles

Disulfide linkage

- MDA-MB-435 cells - PC3 cells

N/A

 PEG triblock polymers showed no interference with the silencing effect of siRNA (39%), in MB-435 cells when compared with unconjugated siRNA  Cellular uptake was significantly reduced in cells treated with 50% molar fraction of siRNA-PEG diblock or triblock copolymer. In contrast, when 1, 5 and 10% molar fraction was used, the levels of uptake were similar to those with no PEG conjugated

[86,167]

HPMA-s-APMA copolymer – siRNA conjugate

Folate

N/A

Disulfide linkage

N/A

 siRNA/folate copolymer was incubated at 37 °C with 5 mM glutathione. Approximately 60% of the siRNA was released within 4 h

[168]

PEG-siRNA conjugates

N/A

Pluronic/ polyethylenimine shell crosslinked nanocapsules with embedded magnetite nanocrystals (PPMC)

Disulfide linkage

- PC3 cells - HeLa, PC3, MDA-MB-435GFP

N/A

 " 3.5 fold greater Cellular uptake (PC-3 cells) on application of an external magnetic field  ; GFP expression levels with the formulation in presence of magnetic field 36.5 ± 13.0% versus 65.9 ± 14.1% in the absence of a magnetic field  PPMCs exhibited no obvious cytotoxicity up to 0.1 mg Fe/ml

[89,90]

6 arm-PEG-siRNA conjugate

Hph1

EGFP

KALA peptide

Disulfide linkage

MDA-MB-435 cells

N/A

 siRNA in 6PEG-siRNA-Hph1-clKALA formulation sustained degradation in 50% serum for 6 h  ; GFP expression level with 6PEG-siRNA-Hph1-clKALA was 31.3 ± 5.6% versus 81.4 ± 1.5% for siRNA/clKALA

[169]

PEG-PLL-PMMAn-MelsiRNA conjugate

N/A

Luciferase

Poly-L-lysine

Disulfide linkage

Neuro2A-eGFPLuc cells

A/J mice

 " Lytic activity of PEG-PLL-PMMAn-Mel-siRNA conjugate, after acidic pre-incubation at pH 5.5 in erythrocytes  ; Luciferase gene expression (50% at 24 nM to >95% at 760 nM) siRNA concentration in vitro  In vivo, conjugates displayed high levels of toxicity with 25 lg of siRNA conjugate being shown to be lethal

[170]

LHRH-PEG-siRNA conjugate

LHRH peptide

VEGF

Poly(ethylenimine)

Disulfide linkage

A2780 cells

N/A

 " Cellular uptake (2 fold) (in vitro) of siRNA conjugate/ PEI complexes containing LHRH as targeting ligand, compared to untargeted siRNA conjugate/PEI complexes  " Gene knockdown in vitro with targeted siRNA-PEGLHRH/PEI compared to untargeted

[171]

PEG-siRNA conjugate

N/A

- KALA peptide - KALA, PEI, PLL - PEI

Disulfide linkage

N/A

 ; (25% and 50%) GFP expression levels with KALA/ siRNA and KALA/PEG-siRNA respectively in MDAMB435 cells  ; (80%) VEGF expression levels in PC-3 cells using KALA/ PEG-VEGF siRNA

[172– 174]

Survivin

- EGFP - VEGF

- GFP - VEGF - VEGF

N/A

- MDA-MB-435GFP cells - PC3 cells - PC3 cells

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Conjugate

332

Table 2 (continued) Target ligand

RNAi gene target

Polymer

Type of conjugation

In vitro

In vivo

Comments

Refs.

Peptide-siRNA and cholesterol-siRNA conjugate

Peptide mimetic of IGF1, D-(CysSer-Lys-Cys)

IRIS1

N/A

Disulfide linkage

MCF7 cells

N/A

 " Receptor specific, cellular uptake and gene knockdown (50%) with Targeted conjugates in MCF-7 cells  ; Cell viability for cells treated with tamoxifen (TAM) and those treated with TAM and targeted conjugate in combination

[108]

Peptide-siRNA and cholesterol-siRNA conjugates

TAT and penetratin peptides

p38 MAP kinase

N/A

Disulfide linkage

L929 cells

BALB/c nude mice

    

At siRNA concentration of 10 lM ;(36%) mRNA level with TAT(48–60)-siRNA ; ( 20%) mRNA level with penetratin-siRNA ; (28%) mRNA level with cholesterol-siRNA TAT (48–60)-siRNA and penetratin-siRNA showed 20– 30% and 47% knockdown of p38 MAP kinase in the mouse lung when administered intratrachealy

[64]

Peptide-siRNA conjugate

Penetratin and transportan

Luciferase and GFP

With or without lipofectamine

Disulfide linkage

COS-7 cells, C166GFP EOMA-GFP cells and CHOAA8-Luc Tet-Off cells

N/A

 ; (75% and 40%) in luciferase gene expression in COS-7 cells with Lipofectamine-mediated transfection of penetratin-siRNA and transportan-siRNA conjugates, respectively  ; (86–97%) in GFP protein levels In EOMA-GFP cells with Penetratin-siRNA and transportan-siRNA  ; (67–80%) in GFP protein levels in C166-GFP cells with Penetratin-siRNA and transportan-siRNA

[61]

HIRMAb and TfRMAb

Luciferase

PEGylated immunoliposomes

Biotin-streptavidin linker

C6, RG-2 and U87 cells

CD344 rats

 ; (79–86%) luciferase gene expression with antibody (HIRMAb)-siRNA conjugate in U87 cells  ; (69 and 81%) luciferase gene expression with antibody (TfRMAb)-siRNA conjugate in a C6 rat glioma model and RG-2 rat glioma model, respectively

[175]

Lactosylated PEGsiRNA conjugate

Lactose

RecQL1

Poly-L-lysine

Acid labile linkage using bthiopropionate

Huh-7 cells (express asialoglycoprotein (receptor – ASPGR)

N/A

 ; ( 20%) in gene expression in monolayer culture with 150 nM of siRNA conjugate. In contrast oligofectAMINE showed a 87% reduction in cell growth. <10% tumor volume inhibition of spheroids with the conjugate, whereas no inhibition observed with oligofectamine  Shorter PLL length showed lower efficacy in growth inhibition of spheroids compared to longer PLL chains  Polyplexes were more efficient in penetrating through the spheroids and inducing high levels of apoptosis, in comparison to lipoplexes

[176,177]

Peptide-siRNA conjugate

TQIENLKEKG – peptide sequence

Alexa555labeled scrambled siRNA and Lamin A/C siRNA (siLAM)

Lipofectamine

N-bromoacetyl terminated peptide conjugated with 30 thiopropyl terminated siRNA

ECV-304 cells

N/A

 ; 3-fold reduction in silencing activity of siLAM, when the peptide was conjugated to either the 30 end of the sense or antisense strand  Both sense strand and antisense strand conjugated to the TQIENLKEKG peptide showed a comparable level of target gene expression in ECV-304 cells; in contrast a mutated form of TQIENLKEKG, which showed no biological activity

[178]

Anti-C5aR1 Abprotamine-C5 siRNA conjugate

Anti-C5aR1 Ab

C5, C5aR1 (CD88)

Protamine

T3TM-Max Conjugation Kit,

RAW cells

Collagen Ab-induced arthritis model in C57 BL/6 WT mice

 ; CDA levels in mice, when treated with C5 and C5AR1 siRNA or a combination 13%, 22% & 58% (in combination)  ; C5 (6%) expression level (in vitro) with Ab-protamineC5 siRNA conjugate  ; CDA level (83%) in mice when treated with conjugate, when compared with scramble siRNA control

[179]

Others Antibody-siRNA conjugate

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Conjugate

Table 2 (continued) Target ligand

RNAi gene target

Polymer

Type of conjugation

In vitro

In vivo

Comments

Refs.

Amphiphilic-siRNA conjugates

N/A

Bcl-2

PAMAM dendrimer

Lipophilic moieties DHA DSA, CHL, coupled to antisense oligonucleotides via amino-hexanol linker Bis aliphatic (bisDHA) conjugate involved a phosphoramidite linker followed by amino hexanol, linker

PC3 cells

N/A

   

; (80%) mRNA levels with DSA conjugate ; (31%) mRNA levels with DHA conjugate ; (44%) mRNA levels with CHL conjugate It was shown that the silencing efficiency of Bcl-2 by AONs decreased as HSA (serum) levels increased-however, the incubation of HSA with a 35 M excess of decanoic acid restored the silencing efficiency of AON

[180]

Aptamer-siRNA conjugates

4-1BB aptamer

Multiple siRNA targets

N/A

Durascribe T7 Transcription kit to generate conjugates

HEK293T, HEPA16, and B16-F10.9 cells

N/A

[181]

GalNAc-conjugatesiRNA

GalNAc

Transthyretin (TTR)

N/A

Phosphodiester linkage

Primary mouse hepatocytes

C57BL/6 mice

 Silencing efficiency was restored if aptamers were conjugated to the sense strand instead of the antisense strand  The introduction of a mutation in the 50 end of the sense strand (C ? U) led to an improvement in the silencing ability of siRNA but not to the same levels as unconjugated siRNA  Silencing efficiency of siRNAs are maintained by choosing siRNA sequences with a low Tm (<50 °C)  It was concluded that sequential covalent conjugation of three simple GalNAc moieties through non-nucleosidic linkers to the 30 -end of oligonucleotides are recognized and taken up by ASGPR  Uptake of trivalent GalNAc/siRNA was comparable that of triantennary GalNAc/siRNA. Trivalent GalNAc/siRNA had similar in vitro and in vivo potency as those observed in triantennary GalNAc conjugates  " Cellular uptake affinity exhibited by the modified GalNAC (three clustered monomeric GalNAc units attached to siRNA either through ribosugar or nucleobase as a trivalent ligand design) specific for ASGPR receptor on hepatocytes

Carbachol-siRNA conjugate

Carbachol

Caspase 3

Lipofectamine

Not provided

ParC5 and HSG cells

N/A

 ; (50%) mRNA level in HSG cells with 8.7 lM of conjugate  In Sjorgen Syndrome, inflammatory cytokines promote apoptosis of cells. HSG cells were transfected with the conjugate and then treated with TNF-a and cycloheximide; there was a 33% reduction in early apoptotic cells and a 25% reduction in late apoptotic cells

[184]

Cholesterol conjugated siRNA

APRPG peptide

EGFP and Luciferase

DCP-TEPA-based polycation liposomes

Not provided

HT1080 A549-lucC8 and B16-F10luc2 and Colon26 NL-17 cells

BALB/c nude mice

 ; (60%) GFP KD with TEPA-PCL/siRNA conjugate (in vitro)  " Biodistribution profile with TEPA-PCL/siRNA conjugate modified with 10% PEG6000  " tumor retention in the colon26NL-17 carcinoma bearing mice, treated with PEG and TEPA-PCL/siRNA conjugate

[185]

[182,183]

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Conjugate

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Fig. 2. Conjugation of PMOs and PNAs to CPPs. (A) PMO is functionalized at the 50 end with a piperazine ring which is conjugated to the CPP using HBTU/HOBT peptide coupling reagents [67]; (B) PNAs are functionalized at the N-terminus with either an a-bromoacetyl group of a cysteine residue to allow conjugation of a CPP with a Cterminal cysteine via either a thioether or a disulfide linkage [79]; (C) A CPP library functionalized at the N-terminus with alkyne groups was used to conjugate a PNA modified with an azide via copper(II)-catalyzed ‘click’ chemistry. The attachment of a biotin tag using a reducible linker to the PNA resulted in a simple purification strategy via attachment to a streptavidin support [80].

(PBAVE), which is effective at disrupting endosomal membranes. The polymer contains lipophilic butyl side chains as well as hydrophilic amino side chains which are modified with either PEG or the hepatocyte-targeting ligand N-acetylgalactosamine (GalNAc) [95]. These additional components are linked via acid-labile maleamate bonds which mask the primary amines while in circulation, but are cleaved in the endosome to reveal the amines which promote endosomolysis. siRNA is also conjugated to the primary amine groups via a disulfide bond which is hydrolyzed in the endosome allowing interaction with RISC. DPCs were shown to specifically target hepatocytes in mice and effectively silence the apoB and ppara genes, resulting in up to 90% knockdown following a

2.5 mg/kg dose [95]. Later studies showed that replacing the polymer with a GalNAc-conjugated endosomolytic peptide (melittinlike peptide) and co-injecting with a cholesterol-siRNA gave much higher efficacy, and was able to efficiently silence hepatitis B viral gene expression in mice and non-human primates [96]. The development of DPCs is currently being undertaken by Arrowhead Research Corporation, with their DPC-based hepatitis B drug ARC-520 currently in Phase 2 clinical trials, and ARC-AAT for Alpha-1 Antitrypsin Deficiency (AATD) to enter Phase 1 trials (www.arrowheadresearch.com) in the near future. Another recent study similar to the PBAVE-siRNA conjugate involved the use of biodegradable poly(amide) polymers instead

M. Gooding et al. / European Journal of Pharmaceutics and Biopharmaceutics 107 (2016) 321–340

of PBAVE. The poly(amide) polymer was synthesized using a Ncarboxy anhydride (NCA) polymerization scheme that was produced by controlled polymerization and yielded less toxicity [97,98]. In addition to the siRNA conjugates formed with polyamine polymers, most other studies in literature utilize cationic polymers as a complexing agent to facilitate the delivery of siRNA-conjugates across the cell membranes. Table 2 specifically lists the most recent studies with siRNA-conjugates, wherein the conjugate is most-preferably developed using a cleavable, disulfide linkage utilizing a cationic polymer as a complexing agent for enhanced delivery. It should be noted however that, several studies engaging the use of cationic polymers, also used a targeting ligand to ensure receptor-specific cellular uptake. 2.4. Targeting ligands – ON conjugates An effective ON therapeutic should not only have good cell uptake characteristics, but also act specifically on the target tissue, thus avoiding off-target effects and reducing the effective dose. One way of achieving tissue selectivity is to conjugate a targeting ligand which binds to cell-specific receptors on the cells surface. As we saw in the case of DPCs, GalNAc is a carbohydrate ligand that targets hepatocytes by binding to the asialoglycoprotein receptor

335

(ASGPR) [99]. Alnylam Pharmaceuticals have developed bi- and trivalent GalNAc ligands which are conjugated to siRNA via solid phase synthesis [100] (Fig. 3A). The resulting conjugates were capable of effective gene silencing in the livers of mice given a subcutaneous administration with an ED50 of 1 mg/kg [100]. Alnylam have several drugs currently in clinical and preclinical trials using this technology, including ALN-PCS for hypercholesterolemia, ALN-TTRsc for Familial Amyloid Polyneuropathy and Familial Amyloid Cardiomyopathy, and ALN-AT3 for hemophilia (www.alnylam.com). Small peptide ligands have also been used as targeting ligands (Table 1). One of the most widely studied ligands in this category is RGD tripeptide (and its cyclic variant, cRGD) which targets aVb3 integrin, which is overexpressed in various tumors [101]. Two studies in which SSOs linked to a bivalent cRGD via a nonlabile thioether linkage, delivered to cells either alone [102] or as a complex with PEI [103], have shown that addition of the peptide leads to changes in intracellular trafficking compared to the unconjugated SSO, suggesting a switch to a receptor-mediated endocytosis mechanism as a result of integrin binding. In another study, siRNA was conjugated to bi-, tri- and tetravalent cRGD via a thioether bond [104] (Fig. 2B). The RGD-conjugated siRNA was selectively taken up by cells overexpressing aVb3 integrin, and

Fig. 3. Examples of small ligands for cell-specific targeting of ONs. (A) Trivalent GalNAc used by Alnylam for targeting to hepatocytes [100]. The ligand is conjugated to the 30 end of the ON siRNA by solid phase synthesis. (B) Trivalent cRGD for targeting to tumor cells [104]. The thiol allows conjugation to the sense strand of a maleimide-modified siRNA.

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addition of free RGD blocked uptake into these cells. It was also found that increased valency of the conjugate led to an increase in luciferase knockdown efficiency, despite no difference in cellular uptake, suggesting a difference in intracellular trafficking depending on receptor binding, for example increased disruption of the endosomal membrane. Following this in vitro study, an in vivo study has now been carried out using an siRNA-cRGD conjugate against vascular endothelial growth factor receptor 2 (VEGFR2), which was injected into a mouse cancer model [105]. The conjugate successfully knocked down the target gene, leading to decreased angiogenesis in the tumors and resulting in a significant reduction in tumor growth. Other peptide targeting ligands that have been conjugated to ONs include nine amino acid bombesin (BBN) peptides, which target the BB2 G-protein coupled receptor overexpressed in cancer cells. This has been used to successfully target an SSO to the nucleus of PC3 prostate cancer cells [106]. In a further study, histidine residues were added to a trivalent BBN conjugate which successfully increased nuclear localization of the ON and increased splice-correcting activity via the proton buffering capabilities of the histidines [107]. An insulin growth factor-1 (IGF-1) peptide mimetic, which targets the IGF-1 receptor, has also been conjugated via a peptide bond to siRNA and was taken up into breast cancer cells producing a knockdown of a target protein comparable to a cholesterol-siRNA conjugate [108]. In addition to the small ligands discussed above, large targeting molecules have also been conjugated to ONs in order to achieve cell- and tissue-specific delivery. Antibodies are a potential targeting molecule which bind to cell surface receptors with high specificity and affinity, and may be easily generated against novel targets. Covalent conjugates of ONs with antibodies are only recently being explored in the literature [109,110], with most studies making use of noncovalent complexes with antibody fusion proteins with protamine [111] and nonaarginine [112] or using biotin-streptavidin coupling [113]. One study compared the nuclear translocation abilities of an antisense DNA linked to a herceptin antibody either in the form of non-covalent biotinstreptavidin nanoparticles, or via thioether covalent conjugation [114]. In both studies, the DNA was delivered to the cytoplasm, but only the DNA delivered as a non-covalent nanoparticle was observed in the nucleus, presumably following dissociation of the particle. Another study compared disulfide conjugated STAT3 siRNA to an anti-Lewis-Y monoclonal antibody with a noncovalent structure formed from an antibody-octaarginine construct and the same siRNA [115]. In this case, the reducible disulfide bond would allow the conjugate to be cleaved in the endosome and therefore the antibody should not interfere with intracellular trafficking of the siRNA. Both constructs specifically entered cells overexpressing the Lewis-Y antigen, but only the non-covalent complex was able to silence the target gene. The covalent conjugate required the addition of either chloroquine or cationic nonaarginine peptide in order to elicit a significant gene silencing effect. These two studies confirm that ON-antibody conjugates are less effective than non-covalent particles due to poor intracellular trafficking, irrespective of whether a labile or non-labile linkage is used. However, a recent study conjugated an antisense ON via a disulfide linkage to an antibody against a-CD19, a biomarker against acute lymphoblastic leukemia (ALL) [116] wherein the conjugate effectively knocked down the target ALL fusion protein both in vitro and in vivo, with a dose of 93 nmol/kg more than doubling the survival time of a human ALL mouse model without the addition of any endosomolytic agents. The reason for this discrepancy is unclear, but it may suggest that endosomal escape of ONantibody conjugates is receptor specific, since the authors point out that the CD19 antibody-receptor complex shows close physical

attachment to the endosomal membrane and hence could trigger endosomal leakage [116]. 3. Conclusion The use of ONs to silence or correct defective genes has the potential to treat a wide range of diseases and has been the subject of an intense research effort over the last two decades. The difficulty in delivering ONs to their intracellular site of action remains a significant challenge, but in the last few years a number of potential vectors have emerged that show great potential for clinical use. Among the many ON conjugates outlined in this review, Alnylam’s GalNAc-targeted siRNA is currently a leading candidate for the first siRNA-based drug to be approved, but competition from rival systems such as Arrowhead’s DPCs means that it remains to be seen whether covalent conjugates or non-covalent nanoparticles will prove to be the best system for delivery of ONs for therapeutic use. Acknowledgments The authors wish to acknowledge the research funding from the Irish Research Council (GOIPD/2014/151), a FRSQ postdoctoral fellowship to MM, and the Irish Cancer Society for a research scholarship to JCE (CRS12EVA). References [1] C.F. Bennett, E.E. Swayze, RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform, Ann. Rev. Pharmacol. Toxicol. 50 (2010) 259–293. [2] G.B. Mulamba, A. Hu, R.F. Azad, K.P. Anderson, D.M. Coen, Human cytomegalovirus mutant with sequence-dependent resistance to the phosphorothioate oligonucleotide fomivirsen (ISIS 2922), Antimicrob. Agents Chemother. 42 (1998) 971–973. [3] S.T. Crooke, R.S. Geary, Clinical pharmacological properties of mipomersen (Kynamro), a second generation antisense inhibitor of apolipoprotein B, Br. J. Clin. Pharmacol. 76 (2013) 269–276. [4] V.K. Sharma, R.K. Sharma, S.K. Singh, Antisense oligonucleotides: modifications and clinical trials, MedChemComm 5 (2014) 1454–1471. [5] M. Malhotra, S. Nambiar, V. Rengaswamy, S. Prakash, Small interfering ribonucleic acid design strategies for effective targeting and gene silencing, Expert Opin. Drug Discov. 6 (2011) 269–289. [6] M.T. Tse, Antisense therapeutics: nuclear RNA more susceptible to knockdown, Nat. Rev. Drug Discovery 11 (2012). 674-674. [7] K. Moelling, F. Broecker, J.E. Kerrigan, RNase H: specificity, mechanisms of action, and antiviral target, Methods Mol. Biol. (Clifton, N.J.) 1087 (2014) 71– 84. [8] S.L. Ameres, P.D. Zamore, Diversifying microRNA sequence and function, Nat. Rev. Mol. Cell Biol. 14 (2013) 475–488. [9] L. He, G.J. Hannon, MicroRNAs: small RNAs with a big role in gene regulation, Nat. Rev. Genet. 5 (2004) 522–531. [10] M.A. Havens, M.L. Hastings, Splice-switching antisense oligonucleotides as therapeutic drugs, Nucleic Acids Res. (2016) [Epub ahead of print]. [11] J. Bauman, N. Jearawiriyapaisarn, R. Kole, Therapeutic potential of spliceswitching oligonucleotides, Oligonucleotides 19 (2009) 1–13. [12] K. Gavrilov, W.M. Saltzman, Therapeutic siRNA: principles, challenges, and strategies, Yale J. Biol. Med. 85 (2012) 187–200. [13] S. Spitzer, F. Eckstein, Inhibition of deoxyribonucleases by phosphorothioate groups in oligodeoxyribonucleotides, Nucleic Acids Res. 16 (1988) 11691– 11704. [14] J. Winkler, M. Stessl, J. Amartey, C.R. Noe, Off-target effects related to the phosphorothioate modification of nucleic acids, ChemMedChem 5 (2010) 1344–1352. [15] M. Majlessi, N.C. Nelson, M.M. Becker, Advantages of 20 -O-methyl oligoribonucleotide probes for detecting RNA targets, Nucleic Acids Res. 26 (1998) 2224–2229. [16] B. Larrouy, C. Boiziau, B. Sproat, J.J. Toulmé, RNase H is responsible for the non-specific inhibition of in vitro translation by 20 -O-alkyl chimeric oligonucleotides: high affinity or selectivity, a dilemma to design antisense oligomers, Nucleic Acids Res. 23 (1995) 3434–3440. [17] M. Gaglione, A. Messere, Recent progress in chemically modified siRNAs, Mini Rev. Med. Chem. 10 (2010) 578–595. [18] J. Kurreck, E. Wyszko, C. Gillen, V.A. Erdmann, Design of antisense oligonucleotides stabilized by locked nucleic acids, Nucleic Acids Res. 30 (2002) 1911–1918. [19] L.F.R. Gebert, M.A.E. Rebhan, S.E.M. Crivelli, R. Denzler, M. Stoffel, J. Hall, Miravirsen (SPC3649) can inhibit the biogenesis of miR-122, Nucleic Acids Res. 42 (2014) 609–621.

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