Peptide-Based In Vivo Delivery Agents for Oligonucleotides and siRNA

Abstract

Introduction

The concept of using discrete peptides as aids to enhance the cell delivery and biological activity of oligonucleotides (ONs), initially through covalent conjugation, can be traced back to 1994 (Bongartz et al., 1994; Allinquant et al., 1995). However, it is only in recent years that significant results have been obtained in experimental animals for peptide-aided ON and small interfering RNA (siRNA) delivery. This perspective focuses on recent reports of in vivo applications, which show that peptide-based delivery is moving closer to clinical use.

The original covalent peptide conjugation concept is now accompanied by newer approaches, notably peptides as complexing agents as well as peptides as cell targeting ligands displayed on nanoparticles and other delivery vectors. Such methodologies offer opportunities to increase the stoichiometric ratio between the peptide delivery agent and the ON or siRNA cargo, which can lead to improved cellular uptake and biological activity and greater protection from cargo nucleic acid degradation during systemic passage, as well as other beneficial pharmacological features. Such features of vector-based approaches have to be balanced against the relative simplicity (and hence lower cost) of covalent peptide–cargo conjugation. Whereas this perspective highlights only the most promising in vivo applications with therapeutic relevance (Table 1), the relative merits of peptide-based approaches on cell uptake and in vivo delivery are debated in greater depth in some recent review articles (Ezzat et al., 2010; Järver et al., 2010; Van den Berg and Dowdy, 2011; Margus et al., 2012).

Table 1.

Structures of Selected Peptides Used for In Vivo Delivery of Oligonucleotides

	Name	Sequence	References
Peptide conjugates	RXR4	RXRRXRRXRRXRXB^(a)	Burrer et al., 2007
			Deas et al., 2007
			Moulton and Moulton, 2008
			Moulton and Moulton, 2010
			Wood, 2010
			Wood et al., 2010
	B peptide	RXRRBRRXRRBRXB^(a)	Du et al., 2011
			Moulton and Moulton, 2008
			Moulton and Moulton, 2010
			Wood, 2010
			Wood et al., 2010
	Pip5e	RXRRBRRXRILFQYRXRBRXRB^(a)	Yin et al., 2011
	B-MSP	RXRRBRRXRRBRXBASSLNIAX^(a)	Yin et al., 2010
	Basic amphipathic peptide	GhRRAFhRRAhRhRhRFhRR^(a)	Wancewicz et al., 2010
	Penetratin	RQIKIWFQNRRMKWKK^(a)	Rogers et al., 2011
	Fragment F3 of (HMG)N2	KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK^(a)	Henke et al., 2008
	SPACE	ACTGSTQHQCG	Hsu and Mitragotri, 2011
Peptide and lipopeptide complexes	PEG-Pep-3	PEG-KWFETWFTEWPKKRK^(b)	Morris et al., 2007
	Chol-MPG-8	Chol-BFLGWLGAWGTMGWSPKKKRK^(b)	Crombez et al., 2009
	CADY	GLWRALWRLLRSLWRLLWRA^(b)	Divita et al., 2009
	Stearyl-TP-10	Stearyl-AGYLLGKINLKALAALAKKIL^(a)	Lehto et al., 2011
	Pepfect 6	Stearyl-AGYLLG[K^*]INLKALAALAKKIL^(a)	El Andaloussi et al., 2011
	PTD-DRBD	GRKKRRQRRRPPQ-[DRBD] (fusion protein)	Michiue et al., 2009
Peptide display on nanovectors	RVG-Arg₉	YTIWMPENPRPGTPCDIFTNSRGKRASNG(R)₉	Alvarez-Erviti et al., 2011
			Kumar et al., 2007
			Pulford et al., 2010
			Son et al., 2011

C-terminal amide.

Cysteamide moiety on the C-terminus.

B, β-alanine; Chol, cholesterol; DRBD, double-stranded RNA binding domain; HMG(N2), high mobility group protein N2; hR, homo-arginine; [K^*], K-K-(K-trifluoromethylquinoline₂)₂; PTD, protein transduction domain; RVG-Arg, rabies virus glycoprotein-arginine; RXR4, (R-Ahx-R)₄Ahx-βAla; SPACE, skin penetrating and cell entering; TP, transportan; X, aminohexanoyl.

Peptide Conjugates

Much of the impetus for exploring peptides as cell delivery agents for ONs has come from the discovery of cell penetrating peptides (CPPs), also known as protein transduction domains (PTDs), such as human immunodeficiency virus (HIV)-1 Tat peptide 48-60 and the Drosophila melanogaster homeobox protein helix 3 peptide (Penetratin) (LANGEL, 2007; 2011). Many other potential CPPs were discovered subsequently or created synthetically, but in vivo applications have been rare, perhaps because of the susceptibility of many CPPs to proteolysis in serum. Further, most CPPs are cationic and thus attempts to conjugate ONs containing phosphodiester or phosphorothioate (PS) linkages often resulted in aggregation and hence insolubility. Even where solubility was achieved, the benefit of CPP attachment often failed to translate into sufficient intracellular activity.

A breakthrough occurred with the realization that CPP conjugation was more suited to charge neutral ON analogues peptide nucleic acids (PNA) and phosphorodiamidate morpholino oligonucleotides (PMO) used in steric blocking antisense applications. Here a new class of arginine (Arg)-rich peptide has been particularly successful in vivo, where Arg residues are spaced by aminohexanoyl and/or β-alanyl units to provide both increased hydrophobicity and a degree of proteolytic resistance in serum, notably (R-Ahx-R)₄Ahx-βAla and (R-Ahx-R-R-βAla-R)₂Ahx-βAla, called RXR4 and B peptides respectively (reviewed in Moulton and Moulton, 2008). First, peptide-PMOs (P-PMOs) were seen to substantially improve antisense activity in vivo over naked PMO, for example in the remarkable protection given to mice upon infection with West Nile virus or coronavirus by RXR4-PMO conjugates targeted to specific viral RNA sites involved in translation or replication (Burrer et al., 2007; Deas et al., 2007). But the company involved (AVI Biopharma) has more recently concentrated instead on developing a newer class of derivative PMO (called PMOPlus™) as antivirals and antibacterials, which has cationic charges along the backbone rather than through an attached peptide (Melbye et al., 2010).

Particular promise was achieved in a series of studies of RXR4-PMO and B-PMO conjugates in a mdx mouse model of Duchenne muscular dystrophy (DMD), which showed dramatically improved dystrophin production following intravenous (i.v.) bolus delivery compared to splice-redirecting naked PMO (reviewed in Moulton and MOULTON, 2010; WOOD, 2010; Wood et al., 2010). Current phase 2b clinical trials with an exon 51 skipping naked PMO (Eteplirsen) in the United States in DMD patients are being carried out at up to 50 mg/kg, following initial dose escalation studies carried out in the United Kingdom to 25 mg/kg that showed variable levels of dystrophin production in muscle biopsy (Cirak et al., 2011). Use of a P-PMO would undoubtedly reduce the dose needed substantially, but in safety studies in monkeys involving bolus injection of B-PMO targeting exon 50, concerns were raised of possible kidney toxicity at higher dosages (Moulton and MOULTON, 2010; www.avibio.com/our-programs/rare-diseases/duchenne-muscular-dystrophy/).

Recent results in mdx mice using PMO conjugated to a similar Arg-rich peptide Pip5e that includes an additional 5-amino acid hydrophobic core showed high dystrophin production upon single 25 mg/kg injection in both skeletal muscle and heart, a significant improvement over B-PMO (Yin et al., 2011). A second lead combines the B-peptide with a muscle-specific peptide (B-MSP-PMO), which showed higher activity in skeletal tissue in the mdx mouse model than B-PMO (Yin et al., 2010). Whether such promise can be translated into a clinical P-PMO lead with a sufficient therapeutic window for DMD treatment remains to be seen. However, such peptide approaches are already starting to be utilized in other ON-targetable neuromuscular and neurodegenerative diseases. For example, a recent report described how a B-PMO, which was active in splicing redirection in a cellular model of ataxia-telangiecatasia, was able to cross the blood–brain barrier and reach Purkinje cells in wild type mice, albeit at high dosage of 60 mg/kg (Du et al., 2011).

There have been numerous reports of peptide conjugates of PNA in antisense applications in vivo, but sadly no application has yet reached the clinic. Recently an Arg-rich amphipathic peptide-PNA conjugate was shown to redirect splicing of PTEN pre-mRNA in adipose tissue of male Balb/c mice by intraperitoneal (i.p.) injection down to a low dose (2.5 mg/kg), demonstrating that precise peptide identity markedly affects PNA tissue distribution (Wancewicz et al., 2010). A new application is the use of triplex-forming PNA conjugated to Penetratin to carry out sequence specific genomic modification of hematopoietic progenitor cells. The peptide was found to slow PNA clearance after i.p. injection, leading to substantial gene mutation rates in multiple cell lineages in somatic tissues, and the results suggest that peptide-PNA may provide new options for treatment of monogenic hematological diseases such as thalassemia (Rogers et al., 2011). ON PNA inhibitors antisense to microRNA (anti-miR) require only a few cationic Lys residues to direct the PNA into the endosomal pathway and block microRNA function without requiring a CPP, as now further shown in vivo in the targeting of miR-155 in mouse spleen (Fabani et al., 2010). The recent discovery that cell uptake of the PNA and resultant targeting of miR-122 in liver cells is also boosted by addition of a terminal thiol moiety (Torres et al., 2011) may stimulate further in vivo studies of PNAs as anti-miRs, especially for tissue types other than liver, which is already well served by more conventional phosphorothioate-based ONs.

An alternative non-CPP type peptide class is finding application in conjugation with negatively charged ON analogues. Such peptides target specific cell types, such as may be over-expressed on cancer cells or other cell types, by binding cell surface receptors as a homing device. In a recent paradigm, a gapmer 2′-O-Me/DNA/PS ON targeting the transcription factor Id1 was coupled to the F-3 31-mer peptide, a fragment of the high mobility group protein (HMG) N2 that homes to neo-vessels in xenograft tumors, and showed substantial inhibition of tumor growth and metastasis in a mouse tumor xenograft model (Henke et al., 2008). The peptide binds to nucleolin, which is over-expressed on the surface of cancer cells and is subsequently transported into the nucleus. It is not clear if this conjugate remains monomeric in solution, or since the peptide is moderately cationic, it may perhaps form nanoparticles or aggregates that provide partial protection against degradation in serum. It is unclear at present whether peptide-based targeting approaches in anticancer therapy will be sufficiently competitive with antibody or aptamer-based conjugate or complex approaches, for example, the exciting application of an aptamer-siRNA conjugate to induce tumor immunity in mice (Pastor et al., 2010).

A cyclic phage-display selected peptide for skin penetration (called SPACE) was conjugated to a synthetic siRNA targeting IL-10 and found to knockdown target protein locally after skin absorption on mouse back (Hsu and Mitragotri, 2011). Further examples of homing peptide delivery of ONs and siRNA into specific tissues are to be expected.

One area that could be investigated more is the use of peptide analogues for delivery of ONs and siRNA in vivo. For example, a retroinverso D-peptide has been used as a conjugate of a splice-redirecting PNA in an in vivo biodistribution study in mice (Maeir et al., 2006). Similarly, an octaguanidine cluster concept has proven useful in vivo as a conjugate of PMO (so called VIVO-morpholinos™), and the recent demonstration of improvement in muscle function for mdx dsytrophin-deficient mice is a very recent example of such use (Widrick et al., 2011). It does not appear that peptoids or other types of peptide analogue guanidinium transporters (Goun et al., 2006) have been used yet as in vivo carriers of ONs or siRNAs.

Peptide and Lipopeptide Complexes

Non-covalent peptide-based approaches to ON and siRNA delivery have recently shown excellent promise in animal models. Such peptides are designed to be used in excess to self-assemble and package the ON or siRNA to form discrete nanoparticles that aid cell uptake. Charge neutralization of the ON is not the only mode of interaction, since hydrophobic packing also appears to play an important role in discrete nanoparticle formation. Interest in this area was stimulated by a report that a PEGylated amphipathic 15-mer peptide (Pep-3) mediated in vivo delivery of an antisense oligonucleotide to cyclin B1 and inhibited prostate carcinoma (PC3) tumor growth following i.v. injection (Morris et al., 2007). Subsequently, a second peptide MPG-8 that was cholesterol functionalized was shown to package siRNA targeting cyclin B1 and also to reduce tumor growth following i.v. injection (Crombez et al., 2009). A third 20-mer peptide (known as CADY) has also shown PC3 tumor growth inhibition when complexed with siRNA (Divita et al., 2009). It is remarkable that CADY-siRNA complexes and others of this peptide class have been shown to enter cells by direct translocation rather than through the endosomal pathway, which is commonly utilized by most other CPPs (Rydström et al., 2011).

Another interesting series of ON and siRNA packaging agents are lipopeptides containing stearic acid tails, which can also interact with nucleic acids to form nanoparticles. Recent success in vivo has come from derivatives of the peptide known as transportan-10 (TP10) (LANGEL, 2007). Stearylated TP10 itself has been shown to form nanoparticles with plasmid DNA and effectively express luciferase following intramuscular injection in mice without inducing inflammatory response (Lehto et al., 2011). A derivative lipopeptide having pH titratable trifluoromethylquinoline moieties attached to side chains to aid endosomal release (known as Pepfect 6) was shown to form nanoparticles with siRNA and knock down HPRT1 mRNA in kidney, lung, and liver of mice upon tail vein infusion at 1 mg/kg (El Andaloussi et al., 2011). A second stearylated peptide variant of TP10 (Pepfect 14) containing pairs of ornithine and leucine residues has shown good potential to deliver exon skipping 2′-O-Me/PS oligos targeted to mutated exon 23 dystrophin in mdx myotubes (Ezzat et al., 2011), but this lipopeptide has not yet found application in vivo.

Another fascinating long peptide that has been used to package siRNA for in vivo delivery was constructed recombinantly from fusion of the HIV Tat PTD peptide with a double-stranded RNA binding domain (DRBD) (known as PTD-DRBD). PTD-DRBD was used to package 2 siRNAs (against epidermal growth factor receptor [EGFR] and Akt20) to induce tumor-specific apoptosis in a glioblastoma model after intracerebral injection, and to also substantially increase mouse survival in this notoriously hard-to-treat cancer type (Michiue et al., 2009). However, no further examples of in vivo delivery have yet been disclosed since the delivery peptide technique was commercialized through the company Traversa.

Peptide Display on Nanovectors

In recent years numerous peptides have been identified by the technique of in vivo phage display that “home” to specific tissues, stem cells, or cancer types (Laakkonen and Vuorinen, 2010; Staquicini et al., 2010) or to cross the blood–brain barrier through receptor-mediated transcytosis (Staquicini et al., 2011). While peptide conjugation is one alternative for delivery of ON and siRNA (Hsu and Mitragotri, 2011), some recent papers have described progress in using such peptides as part of more sophisticated vectors that complex or form nanoparticles with the ON or siRNA as cargo. One peptide in particular from the rabies virus glycoprotein (RVG) that targets acetylcholine receptors has shown particular promise. In the first example, RVG conjugated to Arg₉ complexed with fluorescein isothiocyanate (FITC)-labeled anti-GFP siRNA was shown to be transported to mouse brain after i.v. injection. Anti-SOD1 siRNA administered with RVG-Arg₉ peptide complex was able to reduce protein and mRNA for SOD1 in brain, and siRNA against Japanese encephalitis virus RNA was able to afford robust protection against viral infection in mice (Kumar et al., 2007).

RVG-Arg₉ complexed with siRNA targeting cellular prion protein (PrP^c) showed increased protection against degradation when encapsulated in liposomes and could be delivered to PrP^c-expressing neurons in mouse brain and reduce PrP expression (Pulford et al., 2010). Similarly a plasmid could be delivered to mouse brain by use of complexes of bioreducible disulfide-cross-linked polyethyleneimine to which RVG peptide had been covalently attached (Son et al., 2011). In vivo brain targeting was demonstrated following i.v. injection using an ex vivo fluorescence assay.

Particularly exciting however is the delivery to mouse brain following i.v. injection of exosomes isolated from dendritic cells and encapsulating complexes of RVG-Arg₉ and siRNA to BACE1, a potential therapeutic target in Alzheimer's disease, showing strong target-specific knockdown at both mRNA and protein level (Alvarez-Erviti et al., 2011). Injection of exosomes containing RVG/GAPDH siRNA showed specific delivery into neurons, microglia, and oligodendrocytes. Exosome vectors are in their infancy compared to liposomal or polymer nanoparticles, but this area of research is likely to expand rapidly.

Future Prospects

The use of peptides to deliver ONs and siRNA in vivo has only started to flourish in the last few years. This is partly due to better understanding of how synthetic peptides can be adapted for in vivo use through the use of analogues and also through improved techniques for peptide selection, such as in vivo phage display. Accordingly, many new peptide motifs have emerged with specific properties of cell targeting or with the ability to cross the blood–brain barrier. Significant efforts are now in progress to harness such potential for delivery of ON and siRNA cargoes. Covalent conjugation is of value in favorable cases, but the development of several types of self-assembly peptide vectors as well as the display of targeting peptides on more sophisticated nanoparticle or exosome vectors are now making significant impact in in vivo animal models.

Peptides face many of the same caveats in their use as do targeting antibodies, such as in their potential for stimulation of unwanted immune responses. However, the small size of many peptides and the use of analogues reduce the risk of immune activation somewhat. Peptides have potential also for other toxic side effects in vivo, but sadly very little published information is available on their in vivo toxicology. Arg-rich peptides used as conjugates of PMO are thought to have issues of kidney toxicity in monkey at high dosages (Moulton and MOULTON, 2010), and further clinical development here will depend on careful selection of specific peptide sequences that allow a sufficient therapeutic index. Cationic nanoparticles (whether or not peptidic) also suffer from the potential for kidney toxicity, due mostly to their rapid excretion, as exemplified in a monkey study of cyclodextrin polycation delivery of siRNA (Heidel et al., 2007). Whether such toxicities will limit the use of peptide-based nanovectors remains to be seen.

As mentioned above, peptide ligands compete with selected nucleic acids–based aptamers for cell targeting, which recently have made particularly strong advances in the anticancer field (Pastor et al., 2010). The potential clinical use of receptor-targeted approaches for synthetic nucleic acids is nicely discussed in a recent review (Ogris and WAGNER, 2011). Progress along the clinical pathway will remain dependent on to what extent particular peptides can impart advantages in cell delivery in vivo over antibodies and aptamers, such as in pharmacology, biodistribution, and of course cost. Clearly, the pharmaceutical industry now accepts peptides themselves as drugs and it seems likely that it will not be long before some peptide-mediated ON and siRNA application makes it to the clinic.

Footnotes

Acknowledgments

We thank other members of the Gait group for manuscript reading and comments. This work was supported by the Medical Research Council (MRC reference U105178803). Thibault Coursindel is supported by a grant from the Association Française contre les Myopathies (AFM programme 14784).

Author Disclosure Statement

No competing financial interests exist.

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