Peptide-mediated Cell and In Vivo Delivery of Antisense Oligonucleotides and siRNA

Abstract

Antisense oligonucleotides and short interfering RNAs (siRNAs) are nucleic acids-targeting reagents for gene expression modulation that are being developed as drugs for many applications. A number of useful synthetic nucleic acids analogues have been introduced recently to greatly improve their properties for use as therapeutics. However, their full effectiveness in cells and in vivo has often only been realized through development of suitable nonviral delivery systems. Among these, a range of natural and synthetic peptides have been found useful for enhancing cellular uptake and/or cell targeting of oligonucleotide analogues and siRNA. Such peptides are synthetically conjugated, used as noncovalent complexes, or used in combination with polymer, liposomal or exosome formulation techniques. This review begins by describing the modes of action of antisense reagents and siRNA and goes on to focus on recent advances in their peptide-mediated cell and in vivo delivery and how peptide use has influenced drug development. The review discusses the challenges associated with understanding the physiological and toxicological aspects of peptide-mediated delivery. Developments towards clinical use are also highlighted, with particular emphasis on peptide conjugates of oligonucleotide analogues used for treatment of neuromuscular diseases. Antisense oligonucleotides are used widely to interfere with biological processes in cells and are being developed in some cases as drugs.1Kole R Krainer AR Altman S RNA therapeutics: beyond RNA interference and antisense oligonucleotides.Nat Rev Drug Discov. 2012; 11: 125-140Crossref PubMed Scopus (373) Google Scholar,2Kurreck J Therapeutic Oligonucleotides. Royal Society of Chemistry, Cambridge2008Crossref Google Scholar There are numerous types of oligonucleotide analogue of varying chemistries, as well as short interfering RNAs (siRNAs), and they encompass a range of intracellular targets and modes of action. Common to all oligonucleotide types is the need to be delivered into cells and tissues efficiently in order to carry out their targeting functions. Some types are able to enter certain cell classes in vivo (such as hepatocytes or kidney cells) reasonably efficiently in naked form (e.g., those containing both phosphorothioate (PS) linkages and certain sugar modifications),3Bijsterbosch MK Manoharan M Rump ET De Vrueh RL van Veghel R Tivel KL et al.In vivo fate of phosphorothioate antisense oligodeoxynucleotides: predominant uptake by scavenger receptors on endothelial liver cells.Nucleic Acids Res. 1997; 25: 3290-3296Crossref PubMed Scopus (120) Google Scholar but bio-availability at the right cellular location may still be limited by poor cell trafficking and endosomal entrapment. In other cell types, cell targeting and entry is found to be poor for many naked oligonucleotide types, or their ability to reach the desired tissue is limited. Accordingly, there have been massive efforts to search for new delivery systems that may enhance tissue penetration, improve cell targeting and cell entry, as well as enhance intracellular bioavailability at the desired biological target. The most prevalent delivery systems for oligonucleotides and siRNA are cationic liposomes and other nanoparticle delivery systems, based initially on cell culture studies and later translated into sophisticated multicomponent in vivo vectors.4Kaneda Y Update on non-viral delivery methods for cancer therapy: possibilities of a drug delivery system with anticancer activities beyond delivery as a new therapeutic tool.Expert Opin Drug Deliv. 2010; 7: 1079-1093Crossref PubMed Scopus (33) Google Scholar,5Xu L Anchordoquy T Drug delivery trends in clinical trials and translational medicine: challenges and opportunities in the delivery of nucleic acid-based therapeutics.J Pharm Sci. 2011; 100: 38-52Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar Among the simpler methods that have been utilized to enhance cell delivery of oligonucleotides and siRNA are cationic peptide vectors, often referred to as cell-penetrating peptides (CPPs) or peptide transduction domains (PTDs). The first CPP was introduced in 19946Derossi D Joliot AH Chassaing G Prochiantz A The third helix of the Antennapedia homeodomain translocates through biological membranes.J Biol Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar and since then there has been a continuous stream of new peptide delivery vectors where increased delivery and better pharmacological properties are claimed and a variety of applications demonstrated.7Said Hassane F Saleh AF Abes R Gait MJ Lebleu B Cell penetrating peptides: overview and applications to the delivery of oligonucleotides.Cell Mol Life Sci. 2010; 67: 715-726Crossref PubMed Scopus (0) Google Scholar The cellular entry and intracellular trafficking mechanisms behind peptide-mediated transduction are still not fully elucidated but are reviewed thoroughly elsewhere.8Margus H Padari K Pooga M Cell-penetrating peptides as versatile vehicles for oligonucleotide delivery.Mol Ther. 2012; 20: 525-533Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,9Juliano RL Peptide-oligonucleotide conjugates for the delivery of antisense and siRNA.Curr Opin Mol Ther. 2005; 7: 132-136PubMed Google Scholar,10Nakase I Akita H Kogure K Gräslund A Langel U Harashima H et al.Efficient intracellular delivery of nucleic acid pharmaceuticals using cell-penetrating peptides.Acc Chem Res. 2011; (epub ahead of print)PubMed Google Scholar,11Juliano RL Ming X Nakagawa O Cellular uptake and intracellular trafficking of antisense and siRNA oligonucleotides.Bioconjug Chem. 2012; 23: 147-157Crossref PubMed Scopus (83) Google Scholar In addition to CPPs, there are peptides designed to target to specific cells or tissues (homing peptides).12Laakkonen P Vuorinen K Homing peptides as targeted delivery vehicles.Integr Biol (Camb). 2010; 2: 326-337Crossref PubMed Scopus (0) Google Scholar This review concentrates on recent progress (since 2007) in all of the various peptide-mediated strategies for delivery of antisense oligonucleotides and siRNA into cells in culture and also in in vivo applications, and points to prospects for clinical development. There are several ways that short oligonucleotides and siRNA can affect cellular processes, schematically illustrated in Figure 1. The most obvious approach, originally proposed by Zamecnik and Stevenson,13Zamecnik PC Stephenson ML Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide.Proc Natl Acad Sci USA. 1978; 75: 280-284Crossref PubMed Scopus (0) Google Scholar is antisense binding through Watson-Crick base pairing to messenger RNA (mRNA) to block translational initiation or elongation and thus decrease the levels of the corresponding protein (Figure 1a). Steric blocking of ribosomal elongation is known to be hard to achieve, but blocking of ribosomal initiation can be efficient by targeting the initiator AUG region, the 5′-cap region, or, where appropriate, an internal ribosome entry site. It is also possible to block polyadenylation.14Bennett CF Swayze EE RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform.Annu Rev Pharmacol Toxicol. 2010; 50: 259-293Crossref PubMed Scopus (459) Google Scholar Such targeting requires delivery of oligonucleotides in stoichiometric amount to that of the targeted mRNA present in the cell at a given time. Steric blocking also requires the use of strongly binding oligonucleotide analogues to ensure good competition for the mRNA with the proteins involved in translation or other cellular processes. Similarly to steric-blocking mRNA binding, some oligonucleotides are also capable of binding in triplex mode to certain specific DNA sequences and inhibiting transcription in the nucleus, leading to a decrease in mRNA production,15Mahato RI Cheng K Guntaka RV Modulation of gene expression by antisense and antigene oligodeoxynucleotides and small interfering RNA.Expert Opin Drug Deliv. 2005; 2: 3-28Crossref PubMed Scopus (0) Google Scholar but difficulties in obtaining sufficient potency when targeting chromosomal DNA selectively make gene expression inhibition through triplex formation currently less attractive as a therapeutic intervention mechanism. However, sequence-specific gene modification via triplex formation is a new subject that has recently shown great potential.16Rogers FA Lin SS Hegan DC Krause DS Glazer PM Targeted gene modification of hematopoietic progenitor cells in mice following systemic administration of a PNA-peptide conjugate.Mol Ther. 2012; 20: 109-118Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar A second mechanism involves antisense binding to RNA followed by recruitment and activation of RNase-H, which consequently leads to degradation of the mRNA target (Figure 1b). This mode of action is limited to those oligonucleotides containing a minimum stretch of 6–10 unmodified DNA or PS DNA units (known as gapmers), but outer nucleotides can be more heavily modified to enhance complementary RNA binding. In principle, catalytic turnover allows a lower concentration of oligonucleotides to be used compared to steric-blocking oligonucleotides. RNase-H–dependent antisense has been used very successfully to reduce expression levels of specific proteins, or to inhibit viral replication.17Jepsen JS Wengel J LNA-antisense rivals siRNA for gene silencing.Curr Opin Drug Discov Devel. 2004; 7: 188-194PubMed Google Scholar,18Laxton C Brady K Moschos S Turnpenny P Rawal J Pryde DC et al.Selection, optimization, and pharmacokinetic properties of a novel, potent antiviral locked nucleic acid-based antisense oligomer targeting hepatitis C virus internal ribosome entry site.Antimicrob Agents Chemother. 2011; 55: 3105-3114Crossref PubMed Scopus (0) Google Scholar In the case of RNA interference, one strand of an siRNA duplex (antisense or guide strand) becomes targeted to the mRNA as a result of recruitment by the RNA-induced silencing complex (RISC) cellular machinery and leads subsequently to RNA cleavage (Figure 1c). The siRNA can be targeted to any accessible part of the mRNA, similar to the RNase-H–dependent oligonucleotide targeting method. The sense (passenger) strand may include chemically modified nucleotides in any position, whereas the antisense strand only tolerates a limited range of modified nucleotides in specific locations. Single-stranded oligonucleotide analogues have shown great promise as inhibitors of micro-RNA (miRNA) action by preventing miRNA binding to mRNA that would otherwise trigger the RNA interference machinery and result in mRNA downregulation or degradation (Figure 1d). Interestingly, good levels of intracellular activity have been achieved without requiring CPP-assisted delivery both in cell culture and in vivo (e.g., for peptide nucleic acid (PNA) and locked nucleic acid (LNA)),19Fabani MM Gait MJ miR-122 targeting with LNA/2’-O-methyl oligonucleotide mixmers, peptide nucleic acids (PNA), and PNA-peptide conjugates.RNA. 2008; 14: 336-346Crossref PubMed Scopus (147) Google Scholar,20Torres AG Fabani MM Vigorito E Williams D Al-Obaidi N Wojciechowski F et al.Chemical structure requirements and cellular targeting of microRNA-122 by peptide nucleic acids anti-miRs.Nucleic Acids Res. 2012; 40: 2152-2167Crossref PubMed Scopus (0) Google Scholar in contrast to naked oligonucleotides targeting mRNA, where high concentrations and extended periods are required in cell culture for RNase-H–dependent activity when delivered gymnotically (from Greek: gymnos, meaning naked).21Stein CA Hansen JB Lai J Wu S Voskresenskiy A Høg A et al.Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents.Nucleic Acids Res. 2010; 38: e3Crossref PubMed Scopus (104) Google Scholar Oligonucleotides targeting miRNAs have been shown to rapidly enter cells and interfere with miRNA activity at submicromolar concentrations.19Fabani MM Gait MJ miR-122 targeting with LNA/2’-O-methyl oligonucleotide mixmers, peptide nucleic acids (PNA), and PNA-peptide conjugates.RNA. 2008; 14: 336-346Crossref PubMed Scopus (147) Google Scholar,22Fabani MM Abreu-Goodger C Williams D Lyons PA Torres AG Smith KG et al.Efficient inhibition of miR-155 function in vivo by peptide nucleic acids.Nucleic Acids Res. 2010; 38: 4466-4475Crossref PubMed Scopus (91) Google Scholar Current thinking is that the oligonucleotide may meet the miRNA somewhere within the endosomal pathway,20Torres AG Fabani MM Vigorito E Williams D Al-Obaidi N Wojciechowski F et al.Chemical structure requirements and cellular targeting of microRNA-122 by peptide nucleic acids anti-miRs.Nucleic Acids Res. 2012; 40: 2152-2167Crossref PubMed Scopus (0) Google Scholar and therefore CPP-enhanced endosomal release may be unnecessary, although the generality of this conclusion for all cell types and miRNAs is yet to be established. Inhibition of miRNA is thought to occur by miRNA sequestration rather than by degradation.20Torres AG Fabani MM Vigorito E Williams D Al-Obaidi N Wojciechowski F et al.Chemical structure requirements and cellular targeting of microRNA-122 by peptide nucleic acids anti-miRs.Nucleic Acids Res. 2012; 40: 2152-2167Crossref PubMed Scopus (0) Google Scholar Therapeutic applications of anti-miRs have been recently reviewed.23Gambari R Fabbri E Borgatti M Lampronti I Finotti A Brognara E et al.Targeting microRNAs involved in human diseases: a novel approach for modification of gene expression and drug development.Biochem Pharmacol. 2011; 82: 1416-1429Crossref PubMed Scopus (45) Google Scholar An alternative mechanism has also proven successful, where a short oligonucleotide binds the mRNA to cover the miRNA-binding site to again prevent RISC binding and subsequent mRNA downregulation (Figure 1e).24Staton AA Giraldez AJ Use of target protector morpholinos to analyze the physiological roles of specific miRNA-mRNA pairs in vivo.Nat Protoc. 2011; 6: 2035-2049Crossref PubMed Scopus (35) Google Scholar Another important mode of action of antisense oligonucleotides involves splicing redirection of pre-mRNA in the cell nucleus. An oligonucleotide directed to regions at, or close to, a splice site can mask normal or aberrant splicing events leading either to exon exclusion (Figure 1f) or exon inclusion (Figure 1g). Splicing redirection model systems have been used very successfully to monitor peptide-mediated oligonucleotide delivery.25EL-Andaloussi S Johansson HJ Lundberg P Langel Ü Induction of splice correction by cell-penetrating peptide nucleic acids.J Gene Med. 2006; 8: 1262-1273Crossref PubMed Scopus (85) Google Scholar,26Abes S Moulton H Turner J Clair P Richard JP Iversen P et al.Peptide-based delivery of nucleic acids: design, mechanism of uptake and applications to splice-correcting oligonucleotides.Biochem Soc Trans. 2007; 35 (Pt 1): 53-55Crossref PubMed Scopus (0) Google Scholar In addition, the methodology is already being assessed in the clinic for treatment of neuromuscular and neurodegenerative diseases where targeting of specific exons in mutated dystrophin pre-mRNA with antisense phosphorodiamidate morpholino oligonucleotides (PMO) allows redirection of the splicing pattern to restore a correct reading frame (exon skipping), resulting in the regeneration of functional dystrophin protein expression.27Wood MJ Gait MJ Yin H RNA-targeted splice-correction therapy for neuromuscular disease.Brain. 2010; 133 (Pt 4): 957-972Crossref PubMed Scopus (65) Google Scholar Stimulation of exon inclusion by antisense oligonucleotides is an alternative mechanism being examined for potential treatment for spinal muscular atrophy.28Passini MA Bu J Richards AM Kinnecom C Sardi SP Stanek LM et al.Antisense oligonucleotides delivered to the mouse CNS ameliorate symptoms of severe spinal muscular atrophy.Sci Transl Med. 2011; 3: 72ra18Crossref PubMed Scopus (126) Google Scholar In addition, exon skipping can be applied to wild-type genes to generate out-of-frame transcripts as a method of normal protein downregulation.29Kang JK Malerba A Popplewell L Foster K Dickson G Antisense-induced myostatin exon skipping leads to muscle hypertrophy in mice following octa-guanidine morpholino oligomer treatment.Mol Ther. 2011; 19: 159-164Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar In addition to low bioavailability, the poor stabilities of unmodified DNA and RNA and rapid clearance from the bloodstream have been major drawbacks for use as therapeutics. Thus, many different oligonucleotide analogues have been developed in order to increase stability, RNA binding, and other therapeutically useful properties. A selection of important oligonucleotide analogues that have been involved with peptide-mediated delivery is presented in Figure 2. Each analogue type has particular properties that make them suitable for different applications.2Kurreck J Therapeutic Oligonucleotides. Royal Society of Chemistry, Cambridge2008Crossref Google Scholar,30Eckstein F The versatility of oligonucleotides as potential therapeutics.Expert Opin Biol Ther. 2007; 7: 1021-1034Crossref PubMed Scopus (58) Google Scholar Perhaps the most utilized and well-studied analogue is the PS linkage where a sulfur atom replaces one non-bridging oxygen atom in the phosphate backbone (Figure 2a). The PS linkage can be included in any DNA- or RNA-type oligonucleotide and increases nuclease resistance, whereas in an otherwise unmodified DNA it permits RNase-H activation. Introduction of the PS linkage reduces RNA-target affinity somewhat but leads to enhanced interaction with plasma proteins, thereby decreasing renal clearance rates.31Levin AA A review of the issues in the pharmacokinetics and toxicology of phosphorothioate antisense oligonucleotides.Biochim Biophys Acta. 1999; 1489: 69-84Crossref PubMed Scopus (328) Google Scholar Despite some concerns as to possible toxic side effects at higher concentrations, PS linkages are included in all FDA-approved oligonucleotides to date, which unfortunately are currently very few.32Sanghvi YS A status update of modified oligonucleotides for chemotherapeutics applications.Curr Protoc Nucleic Acid Chem. 2011; (Chapter 4): Unit 4.1.1-Unit 4.122Crossref Scopus (18) Google Scholar,33Watts JK Corey DR Clinical status of duplex RNA.Bioorg Med Chem Lett. 2010; 20: 3203-3207Crossref PubMed Scopus (0) Google Scholar The 2′-O-methyl nucleotide (2′-OMe), in which a methyl group replaces a hydrogen atom in the 2′-hydroxyl group in the ribose ring of RNA (Figure 2b), also imparts nuclease resistance, but does not permit RNase-H activation. Such non-RNase-H inducing oligonucleotides are particularly valuable in certain applications where the target RNA needs to remain intact (e.g., splicing redirection). Although the 2′-OMe modification is insensitive to endonucleases, it is still partially susceptible to exonuclease degradation. By combining PS linkages and 2′-OMe nucleotides (PS-2′-OMe), much greater in vivo stability has been achieved resulting in several successful applications. Further enhancements of therapeutic activity have been achieved in some cases by including other 2′-O-alkylated nucleotides (such as 2′-O-methoxyethyl, MOE) or 2′-fluoro-2′-deoxynucleotides, resulting in “mixmers”. All these analogues are also used in the wings of gapmers, as well as in particular positions of siRNA. A highly successful nucleotide analogue is LNA, which is a constrained RNA analogue having a methylene bridge between the 2′ and 4′ positions in the ribose ring (Figure 2c). LNA is also unable to activate RNase-H, but due to the constrained backbone LNA has a very high affinity for single-stranded DNA/RNA compared to other analogues. In addition to high affinity, LNAs display high in vivo stability and slower renal clearance, although in rare cases hepatotoxicity has been observed.34Swayze EE Siwkowski AM Wancewicz EV Migawa MT Wyrzykiewicz TK Hung G et al.Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals.Nucleic Acids Res. 2007; 35: 687-700Crossref PubMed Scopus (132) Google Scholar The increased affinity allows LNA to be used in much shorter oligonucleotide than for many other analogue types (tiny LNAs,).35Obad S dos Santos CO Petri A Heidenblad M Broom O Ruse C et al.Silencing of microRNA families by seed-targeting tiny LNAs.Nat Genet. 2011; 43: 371-378Crossref PubMed Scopus (336) Google Scholar However, nonspecific binding of longer sequences of LNAs, which could result in off-target effects, is alleviated by using LNA in combination with unmodified DNA or with other analogues, such as 2′-OMe, in steric-blocking applications. LNA is also used in RNase-H–dependent applications in the wings of all PS gapmers where the central section is PS-DNA. A separate class of analogues are charge neutral. In PMO, the ribose is replaced by a morpholino moiety and the natural phosphodiester linkage is replaced by an uncharged phosphorodiamidate backbone (Figure 2d). The constrained structure and the lack of electrostatic repulsion between the oligonucleotide and its target both contribute to make PMO–RNA interactions more stable compared to DNA–RNA interactions. PMO does not activate RNase-H, but it has been used successfully for steric block antisense purposes, in many in vivo applications and in the clinic.36Moulton HM Moulton JD Antisense morpholino oligomers and their peptide conjugates.in: Kurreck J Therapeutic Oligonucleotides. Royal Society of Chemistry, Cambridge, UK. pp. 43–792008Crossref Google Scholar,37Cirak S Arechavala-Gomeza V Guglieri M Feng L Torelli S Anthony K et al.Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study.Lancet. 2011; 378: 595-605Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar Like PMO, PNA is an uncharged oligonucleotide analogue, where the sugar-phosphate backbone is replaced by repeating N-(2-aminoethyl)-glycine units linked together by amide bonds (Figure 2e). PNA has very high affinity for DNA/RNA that, like PMO, is due partly to the lack of charge repulsion of the peptide-like backbone. PNAs display high selectivity and mismatch discrimination towards its target strand and have been used effectively in many antisense applications and in vivo.16Rogers FA Lin SS Hegan DC Krause DS Glazer PM Targeted gene modification of hematopoietic progenitor cells in mice following systemic administration of a PNA-peptide conjugate.Mol Ther. 2012; 20: 109-118Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar,38Pandey VN Upadhyay A Chaubey B Prospects for antisense peptide nucleic acid (PNA) therapies for HIV.Expert Opin Biol Ther. 2009; 9: 975-989Crossref PubMed Scopus (0) Google Scholar Further, PNA containing a few additional cationic residues (e.g., lysine) have been used successfully to block miRNA activity without the use of a transfection agent or delivery peptide.19Fabani MM Gait MJ miR-122 targeting with LNA/2’-O-methyl oligonucleotide mixmers, peptide nucleic acids (PNA), and PNA-peptide conjugates.RNA. 2008; 14: 336-346Crossref PubMed Scopus (147) Google Scholar,20Torres AG Fabani MM Vigorito E Williams D Al-Obaidi N Wojciechowski F et al.Chemical structure requirements and cellular targeting of microRNA-122 by peptide nucleic acids anti-miRs.Nucleic Acids Res. 2012; 40: 2152-2167Crossref PubMed Scopus (0) Google Scholar The need for improved oligonucleotide therapies has accelerated advances in chemistry and many newer analogues have emerged recently. Unfortunately very few have been able to attain high stability, affinity, and selectivity, while maintaining low toxicity and off-target effects and which are available to researchers in sufficient quantities and reliability for in vivo studies. The analogues selected for mention in this review are those that not only have most of these above attributes but also have been most utilized in peptide-mediated delivery. There are two main ways of utilizing peptide vectors for the delivery of antisense oligonucleotides and siRNA. The first method involves chemical synthesis of peptides that are modified to enable covalent conjugation to oligonucleotides or other cargo types (Figure 3a). Such conjugation reactions may be carried out either on solid support or in solution, and often result in high yields. An advantage of covalent conjugation is that this results in a well-defined single entity that simplifies drug development. Linker types may be chemically stable, which ensures that the peptide–oligonucleotide conjugate remains intact throughout in vivo administration and the subsequent delivery process and that degradation now reflects only the proteolytic susceptibility of the particular peptide. There are numerous conjugation strategies, including thioether, thiol-maleimide, ester formation, and “click” chemistry (reviewed in ref. 39Singh Y Murat P Defrancq E Recent developments in oligonucleotide conjugation.Chem Soc Rev. 2010; 39: 2054-2070Crossref PubMed Scopus (0) Google Scholar,40Lu K Duan QP Ma L Zhao DX Chemical strategies for the synthesis of peptide-oligonucleotide conjugates.Bioconjug Chem. 2010; 21: 187-202Crossref PubMed Scopus (0) Google Scholar). Alternatively, a labile linker cleavable within the cell, such as a disulphide linkage, has proven very successful in studies of peptide-mediated delivery, both in cells and in vivo.41Pooga M Soomets U Hällbrink M Valkna A Saar K Rezaei K et al.Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo.Nat Biotechnol. 1998; 16: 857-861Crossref PubMed Google Scholar,42Lundin P Johansson H Guterstam P Holm T Hansen M Langel U et al.Distinct uptake routes of cell-penetrating peptide conjugates.Bioconjug Chem. 2008; 19: 2535-2542Crossref PubMed Scopus (115) Google Scholar Such disulphide bridges may remain sufficiently stable, if administration is rapid, but may be cleaved later when the conjugate reaches the reducing environment of the endosome/cytoplasm. This approach was initially favored in therapeutic design, because cleavage diminishes the risk of any detrimental effect of the peptide on the interaction of the oligonucleotide with its target. However, recent in vivo applications using stable linkages suggest that peptide-cargo cleavage is not essential. Recent studies have suggested also that thiol or disulfide groups might enhance cellular uptake, although the reasons for this are not as yet fully understood.43Torres AG Gait MJ Exploiting cell surface thiols to enhance cellular uptake.Trends Biotechnol. 2012; 30: 185-190Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar,44Ezzat K Helmfors H Tudoran O Juks C Lindberg S Padari K et al.Scavenger receptor-mediated uptake of cell-penetrating peptide nanocomplexes with oligonucleotides.FASEB J. 2012; 26: 1172-1180Crossref PubMed Scopus (0) Google Scholar Although covalent conjugation is very suitable for charge-neutral oligonucleotide analogues, there are technical difficulties in conjugation and purification of conjugates of highly cationic peptides with negatively charged oligonucleotides that have limited the type of peptide that can be conjugated.45Turner JJ Williams D Owen D Gait MJ Disulfide conjugation of peptides to oligonucleotides and their analogues.Curr Protocols Nucleic Acids Chemistry. 2005; : 4.28.21-24.28.21Google Scholar Sadly, meaningful comparative data on the effects of different peptide-oligonucleotide linkages in biological antisense assays are mostly lacking. The second method of peptide-mediated oligonucleotide delivery exploits the complexing properties of CPPs and their derivatives (Figure 3b). For example, cationic CPPs can form complexes efficiently with negatively charged oligonucleotides.8Margus H Padari K Pooga M Cell-penetrating peptides as versatile vehicles for oligonucleotide delivery.Mol Ther. 2012; 20: 525-533Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar Further, some peptide vectors also contain hydrophobic elements (hydrophobic amino acids or addition of hydrophobic amino acids and/or other moieties such as fatty acids, lipids or cholesterol), which are designed for complex formation with both negatively charged and charge-neutral analogue types. Such complexing peptides form nanosized particles together with the oligonucleotides that are better able to translocate across the plasma membrane (in some cases perhaps avoiding the endosomal uptake system altogether)46Konate K Crombez L Deshayes S Decaffmeyer M Thomas A Brasseur R et al.Insight into the cellular uptake mechanism of a secondary amphipathic cell-penetrating peptide for siRNA delivery.Biochemistry. 2010; 49: 3393-3402Crossref PubMed Scopus (45) Google Scholar and can deliver the oligonucleotide cargo to the target with greater efficiency. It is common to optimize empirically the ratio of the peptide over cargo to obtain the best cellular delivery. The noncovalent strategy does not require the oligonucleotide to be end-modified for conjugation, and thus complex formation is often achieved by simple mixing. Complex formation also results in protection of the oligonucleotide or siRNA from nuclease degradation. On the other hand, structures of varying and undefined size may be generated, which complicates drug characterization and subsequent in vivo use. In the cases of uncharged PMO or PNA, it has been common to first hybridize to a complementary DNA strand (leash) before complex formation with cationic peptide vectors.47Rasmussen FW Bendifallah N Zachar V Shiraishi T Fink T Ebbesen P et al.Evaluation of trans