Review

Cationic PTD/CPP-mediated macromolecular delivery: charging into the cell 1.

Introduction

2.

Arginine-rich PTDs/CPPs

3.

Arginine-rich PTD/ CPP-mediated delivery in vitro and in vivo

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4.

Expert opinion

Peter L€onn & Steven F Dowdy† †

UCSD School of Medicine, Department of Cellular and Molecular Medicine, CA, USA

Introduction: Macromolecular therapeutics, including enzymes, transcription factors, siRNAs, peptides and large synthetic molecules, can potentially be used to treat human diseases by targeting intracellular molecular pathways and modulating biological responses. However, large macromolecules have no ability to enter cells and require delivery vehicles. Protein transduction domains (PTDs), also known as cell-penetrating peptides (CPPs), are a diverse class of peptides that can deliver macromolecules into cells. Areas covered: In this review, we cover the uptake and usage of arginine-rich PTDs/CPPs (TAT-PTD, Penetratin/Antp and 8R). We review the endocytosismediated uptake of these peptides and highlight three important steps: i) cell association; ii) internalization and iii) endosomal escape. We also discuss the array of different cargos that have been delivered by cationic PTDs/CPPs as well as cellular processes and biological responses that have been modulated. Expert opinion: PTDs/CPPs have shown great potential to deliver otherwise undeliverable macromolecular therapeutics into cells for experimentation in cell culture and in animal disease models in vivo. Moreover, over 25 clinical trials have been performed predominantly using the TAT-PTD. However, more work is still needed. Endosomal escape and target-cell specificity remain two of the major future challenges. Keywords: CPP, delivery, macromolecular therapeutics, PTD Expert Opin. Drug Deliv. [Early Online]

1.

Introduction

Macromolecular compounds, such as oligonucleotides, polypeptides and proteins, possess therapeutic features and capabilities that are highly sought after [1,2]. This includes the potential to: i) target specific intracellular molecules, biological pathways and cellular responses with high efficiency and sensitivity; ii) positively or negatively modulate responses by altering specific molecular activities in cells; iii) amplify and potentiate intracellular effects by carrying intrinsic enzymatic activities or by interacting with intracellular enzymes, such as polymerases, kinases or nucleases and iv) change or adapt the modular macromolecular structures of polypeptides and oligonucleotides to enhance function or to circumvent possible resistance mechanisms or desensitization. Collectively, the prospect of utilizing transcription factors, enzymes, peptides, small interfering RNAs (siRNAs) and oligonucleotides to customize treatments against diseases and malignancies is very attractive. However, the major drawback of macromolecules is that they have low to no bioavailability and require delivery vehicles to enter cells [1,2]. Although multiple classes of delivery vehicles have been developed and significant progress has been made, safe and robust uptake formulations are unfortunately still lacking. This remains a severe hindrance for the use of these macromolecules as potent drugs. Consequently, the search for dramatically improved delivery vehicles to harness the unique capabilities

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Cationic PTDs/CPPs, such as TAT-PTD, 8R and Penetratin/Antp, have been used over recent years to deliver a multitude of macromolecular therapeutics in vitro and in vivo. The major route of uptake for cationic PTDs/CPPs is via endocytosis. Still many of the molecular details of this uptake remain unknown. The peptide composition in terms of charged residues and hydrophobic residues not only influences the uptake capability, but also the cellular toxicity. In addition, the cargo may also impact the behavior of the PTD/CPP. Interestingly, while both arginine and lysine have positively charged side groups, the bidentate guanidinium group of arginine has been shown by several groups to be vital for efficient uptake. Endosomal escape is one of the major bottlenecks of PTD/CPP-mediated uptake. Finding more efficient and non-toxic endosomal escape domains is of paramount importance and would likely advance the therapeutic possibilities of PTDs/CPPs dramatically.

This box summarizes key points contained in the article.

of macromolecules as intracellular therapeutics remains a high priority in the field of molecular medicine. One promising approach to deliver macromolecules into cells is by use of peptide/protein transduction domains (PTDs), also described in the literature as cell-penetrating peptides (CPPs) [1,3-9]. PTDs/CPPs surfaced in the late 1980s when two labs, Pabo’s and Green’s, first published data on the ability of the 86 amino acid long HIV protein Trans-Activator of Transcription (TAT) to translocate into cells and modulate transcription [10,11]. Several years after this discovery, a second polypeptide, a 60-amino acid stretch of the Antennapedia homeobox protein (Antp) of Drosophila melanogaster, was also found to be taken up and delivered into cells [12]. Soon afterward, it was discovered that covalent attachment of other proteins and molecules to TAT could piggyback with TAT into cells [13-15]. These intriguing and highly promising results initiated a more detailed characterization of TAT-mediated uptake, which led to the determination of a minimal TAT-PTD domain responsible for transduction [14,15]. Truncating the TAT protein into shorter versions determined that a short positively charged, cationic and arginine-rich nine amino acid peptide was the major contributor to transduction [14,15]. This finding was an important advancement for the usability of TAT-PTDs as it made both the synthesis and size more manageable. Early reports gave an indication of the wide delivery capacity of TAT-PTD by showing uptake of both large- and smallsized cargos into cells in culture and animal models, including uptake of macromolecules such as b-galactosidase, p16 and fluorescein [13-16]. A significant finding at this stage was the evidence that large enzymes could be delivered in vivo, with high efficiency and with preserved enzymatic function [16]. 2

In the aftermath of these findings, many additional PTDs/ CPPs were uncovered and characterized and the field swiftly expanded [1,3-9]. Today, there are more than 100 different PTD/CPP variants described in the literature [3,5] with thousands of papers published on the topic. Furthermore, a multitude of cargo has been successfully delivered by PTDs/CPPs into various cells and in vivo, models, comprising everything from polypeptides to proteins to DNA and RNA to an array of chemical compounds [1,3-9]. Clinical data on PTD/CPP delivery of macromolecules have emerged with over 25 completed Phase I and Phase II clinical trials evaluating the safety and efficacy of PTDs/CPPs in vivo. Backed-up by statistically significant results from Phase II studies, there is currently an ongoing Phase III clinical trial utilizing a TAT-based PTD/ CPP-mediated delivery protocol [17]. Thus, combining PTDs/CPPs with macromolecular drug designs is on the verge of equipping us with a highly adaptable toolbox to potentially treat cancer, viral infections and other diseases. There is now a plethora of published PTDs/CPPs with different properties and presumed routes of uptake [3,4]. However, there are common traits shared between the larger groups of PTD/CPP sequences. For example, positively charged amino acids, such as arginine and lysine, and hydrophobic residues, such as tryptophan and phenylalanine are frequently utilized in multiple PTD/CPP designs. These residues have, in various ways, been shown to promote or facilitate aspects of PTD/CPP-mediated delivery, including cell association, endocytosis, internalization, uptake and/or endosomal escape [1,4,18-26]. In this review, we specifically focus on the progress and usage of arginine-containing PTDs/CPPs to target intracellular molecular pathways using peptides, proteins or oligonucleotides as cargo. 2.

Arginine-rich PTDs/CPPs

Arginine is a well-documented factor of PTDs/CPPs that plays important roles for uptake. Most commonly used arginine-containing PTDs/CPPs are the TAT-PTD (6 arginine residues, RKKRRQRRR), Penetratin/Antp (3 arginine residues, RQIKIWFQNRRMKWKK) and various artificial polyarginine peptides of different lengths, 6 -- 12 moieties (6R -- 12R), with 8R being the most common [3,4,19,26]. These peptides have garnered the lion’s share in the PTD/CPP literature to deliver various molecules and are also involved in a majority of animal models and clinical trials [1,4,7,16,18-26]. Although early on in the field there was much debate surrounding uptake mechanisms of PTDs/CPPs into cells, for arginine-rich PTDs/CPPs, starting in 2004 there has been an ever growing body of high quality data showing that endocytotic pathways, which are well-documented paths of entry into cells [27-29], constitutes the major uptake route [1,4,18-21,24-26,30]. To deliver macromolecular cargo into cells via endocytosis, arginine-rich PTDs/CPPs have to efficiently fulfill three critical steps in a non-cytotoxic fashion (Figure 1). First, they need to associate with the cell surface.

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Cationic PTD/CPP-mediated macromolecular delivery: charging into the cell

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Figure 1. Overview of macropinocytotic uptake of cationic PTDs/CPPs. Cationic PTDs/CPPs bind to negatively charged molecules on the cell surface (a) which triggers Rac1-dependent act in reorganization and macropinocytotic uptake (b), and finally the peptides need to escape from endosomes into the cytoplasm (c). PTDs: Protein transduction domains; CPPs: Cell-penetrating peptides.

Second, they must stimulate and/or undergo efficient internalization by endocytosis. Third, the PTD/CPP cargo has to escape across the lipid bilayer of endosomal vesicles and into the cytoplasm of the cell, which remains the rate-limiting step. Successful passage through this triple-layer endocytosis gauntlet is required for PTDs/CPPs to effectively and efficiently deliver their macromolecular cargo into cells. Arginine-rich PTDs/CPPs and cell association The initial step of cationic PTD/CPP-mediated delivery is the attachment of the transduction peptide, and hence cargo, to the outside of the cell (Figure 1a). This step has been extensively studied over the years [1,4,9,19,21,25,26,30-34]. Electrostatic interactions are formed between the positively charged amino acids of the PTD/CPP and negatively charged molecules at the cell surface. Arginine and lysine are the main contributors to the positive charges of PTDs/CPPs at neutral pH [4,18,19,21,35]. Both have been shown to promote cellular attachments with high efficiency and the number of charges 2.1

per peptides correlates with the interaction and uptake [26]. Too few charges will result in poor cell association and low delivery, while too many will lead to toxicity. The most commonly used cationic PTDs/CPPs are between 8 and 20 amino acids long and possess somewhere between 5 and 8 positively charged residues in different configurations [3,4]. Interestingly, exposing cells to trypsin, which cleaves most of the cell surface proteins, has been shown to abrogate the uptake of TAT-PTD and emphasizes the importance of cell surface protein interactions for proper initiation of transduction [30]. Although both lysine and arginine residues are positively charged and able to promote cell association, the arginine side chains, in contrast to lysine, possess bidentate guanidinium-positive charges that are able to establish hydrogen bonded ion pairs [35]. This is believed to establish interactions with bidentate anionic phosphates, carboxylic acids and sulfates that are present on plasma membrane proteins, lipids and sugars. Interestingly, chemically limiting the arginine R-group charge by methylation to form only a monodentate

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€ nn & S. F. Dowdy P. Lo

bond significantly reduced uptake [35]. This indicates that in addition to a generalized cell association due to charge, there are specific bidentate-dependent arginine-rich PTD/CPP binding sites or partners that are mediating essential roles when it comes to converting cell association into uptake that monodentate lysine does not. Known binding partners of positively charged PTDs/CPPs includes proteoglycans that are negatively charged, abundant and ubiquitously present on most, if not all, cell surfaces, especially sulfated proteoglycans such as heparan sulfate [19,21,30-34]. In addition, overexpression of syndecans, transmembrane proteoglycans, was shown to positively affect uptake of TAT-PTD, 8R and Penetratin/ Antp [33,34]. The universally expressed syndecan-4 isoform displayed the most potent effects. However, the exact roles of proteoglycans in transduction remains unclear and experiments using genetic knockout cell lines have shown that heparan sulfate is not an absolute requirement for protein transduction [30]. In addition to positively charged amino acids, other residues can aid with cell surface association. Hydrophobic residues, such as tryptophan and phenylalanine, have also been shown to contribute to cell association and uptake, potentially by burying their lipophilic R-groups into the lipid bilayer [20,23]. The two tryptophans in Penetratin/Antp [12,33] have long been implicated as a requirement for efficient cell association and transduction. Likewise, the addition of specific protein binding motifs and tumor/tissue homing peptides, such as the integrin-specific arginine--glycine--aspartic acid (RGD) peptide can also be used to help anchor to specific surface molecules [36-38]. These domains, and others, can potentially be used to attract peptides to specific cell types, tissues or sites of disease. So far, there have been few attempts made to combine PTDs/CPPs with RGDs, but in other fields of drug delivery the RGD motif has been more widely investigated [37,38]. In summary, the cell surface targets of PTDs/CPPs remains surprisingly unknown, with none to few actual candidates identified. Further experiments are required and highly warranted in order to delineate the exact direct or indirect roles that proteoglycans play in cationic PTD/CPP-mediated cell association and uptake.

efficiently and instead spread throughout the cell during the fixation process, resulting in an artifactual redistribution throughout the cell [39]. In contrast, live cell microscopy studies of dye-labeled PTDs/CPPs shows that the vast majority of the PTD/CPP is present in punctate spots, endosomes, in the cytoplasm. Consequently, the best studies on PTD/CPP transduction mechanism(s) involve the use of phenotypic assays on live cells that excludes apoptosis or necrosis as there are numerous ways to unintentionally kill cells. The widely different hypotheses on the transduction mechanism(s) likely reflect the multifaceted characteristics of different PTDs/CPPs as well as attachment to different cargos, lab-to-lab experimental conditions and assays. However, for cationic PTDs/CPPs, a more uniform picture emerged starting back in 2004 where a growing body of studies, including ours and others, reported the vital role of energy-dependent, macropinocytosis, a specialized form of endocytosis that most, if not all, cells perform as the mechanism behind the cellular uptake of TAT-PTD, 8R and Penetratin/Antp [18,21,24-26,30,40-45]. The supporting results includes many different assays and conditions as well as data from functional phenotypic assays, such as TAT-Cre-dependent genetic reconstitution of GFP expression and splice-correction assays [24,25,30,45,46]. The strength of these functional assays is that they only mediate a specific read-out if, and only if, the PTDs/CPPs managed to deliver their cargo into the cytoplasm/nucleus of viable cells. Thus, phenotypic transduction assays (excluding cell death) avoid potential false signals from peptides that remain stuck on the outside of cells or are trapped in endosomes. Furthermore, by multiple accounts, cationic PTDs/CPPs are also able to stimulate their own uptake by increasing the baseline rate of macropinocytosis [18,24,25,30]. Although the exact molecular details of this active uptake are still not fully elucidated, PTD/CPP-mediated macropinocytosis is dependent on Rac1-induced act in reorganization [24-26,30,41]. Collectively, these reports point towards a model whereby cell-associated cationic PTDs/ CPPs stimulates macropinocytotic uptake by activating pathways leading to Rac1-dependent act in reorganization and formation of macropinosomes (Figure 1b). Arginine-rich PTDs/CPPs and endosomal escape The rate-limiting step of cationic PTD/CPP transduction is escape of the PTD/CPP-cargo from endosomes into the cytoplasm and nucleus of cells (Figure 1c). Given the plethora of studies that display functionally relevant and well-controlled phenotypes after PTD/CPP addition, it is well documented that some fraction of PTD/CPP-cargo is escaping into the cytoplasm and nucleus of cells. Although the mechanistic details remain unknown, one thing is clear, endosomolytic capabilities of cationic PTDs/CPPs remains the major bottleneck for delivery of macromolecules into cells [47]. Indeed, live cell microscopic studies of dye-labeled PTDs/CPPs display punctate patterns in the cytoplasm demonstrating that the vast majority of PTD/CPP-cargo remains stuck in endosomes 2.3

2.2

Arginine-rich PTDs/CPPs and endocytosis

The second phase of transduction is to progress from cell association to internalization via endocytosis (Figure 1b). Despite the fact that a plethora of reports have been generated on the basis of this topic, the actual mechanism(s) of uptake for the bulk of PTDs/CPPs are still not very well resolved. Over the last 20 years, different routes of entry have been proposed, ranging from direct cell membrane penetration to various forms of endocytosis [3,4,9]. Due to the strong cell surface association of PTDs/CPPs, we note that previously there have been many false-positive artifacts observed in cells that were fixed after treatment with dye-labeled PTDs/CPPs. For unknown reasons, cationic PTDs/CPPs do not fix very 4

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Cationic PTD/CPP-mediated macromolecular delivery: charging into the cell

with only a very small fraction able to escape [47,48]. Furthermore, it remains unknown exactly when and how that small portion manages to escape. In our hands using different phenotypic assays, we guesstimate that a fraction of 1% escapes into the cytoplasm. Although important efforts have already been made, finding novel ways of enhancing endosomal escape is of paramount importance. The vast majority of endosomal escape efforts take their cue from the mechanisms that enveloped viruses use to escape endosomes during infection [49]. The pH of endosomes drops from the extracellular pH of ~ 7.2 to ~ 5 [50]. Both influenza and Ebola viruses express pH-sensitive, hydrophobic peptide motifs on their surface that insert into the endosomal membrane in low pH-dependent manner resulting in membrane destabilization that drives fusion of the viral envelope membrane to the endosomal membrane resulting in release of viral capsid into the cytoplasm of the cell [49]. Building on this knowledge, we have attached the influenza HA2 fusogenic domain to the TAT-PTD resulting in a pH-dependent significantly improved transduction of Cre recombinase into cells [24]. Likewise, hydrophobic amino acids, such as tryptophan and phenylalanine, have also been shown to significantly aid with release of PTDs/CPPs [20,22,23]. Here, the R-group side chains of the hydrophobic residues are thought to bury themselves into the lipid bilayer causing membrane destabilization. Collectively, the trick learned from the enveloped viruses is to incorporate a pHdependency so that the membrane destabilization happens only in the endosome and not on the cell surface, which could lead to unintentional cell death. However, with an exact amount of hydrophobicity, it may still be possible to maintain an intact plasma membrane while, once the peptides are densely packed into endosomes, causing enough instability to efficiently disrupt endosomal vesicles. Multivalent PTDs/CPPs is another approach that has been shown to aid endosomal escape by increasing local concentration of PTDs/CPPs [47]. However, this approach has a steep cytotoxicity dose curve. An alternative approach that has long been touted for nanoparticle escape from endosomes is to take advantage of the proton sponge effect [51]. Use of histidine residues that have a pKa of ~ 6 attached to CPPs/PTDs have been another way of generating endosomolytic function after acidification of endosomal vesicles [47,52]. The endosomolytic endoporter peptide combines both the putative proton sponge effect of histidine residues with hydrophobic leucine residues to enhance endosomal escape [53]. The success of the approach, as with all proton sponge approaches, appears to require an excessively high concentration of the endosomolytic agent. In addition, other lysosomotropic agents, such as chloroquine or trifluoromethylquinoline, are frequently co-incubated or directly attached to PTDs/CPPs to significantly aid with escape into the cytoplasm [4,24,54,55]. However, many of the methods suggested in this section also run the risk of increased toxicity. Both chloroquine and endoporter show steep cytotoxicity dose curves. So while these

molecules lay a clear path to the importance of enhancing endosomal escape, finding new ways to harness improved endosomal escape without increasing cell toxicity will be one of the great challenges of the future of PTD/CPP-mediated macromolecular delivery.

Arginine-rich PTD/CPP-mediated delivery in vitro and in vivo

3.

Molecular cell biology has advanced tremendously over the last couple of decades and remarkable details have been uncovered in regards to how molecular pathways governor are altered in disease. Thus, there is great potential and hopes of turning this broad molecular knowledge into useful therapeutic opportunities. Unfortunately, many therapeutic approaches to target molecular pathways are undruggable with traditional small molecule therapeutics and require macromolecular therapeutics, including recombinant proteins, transcription factors, enzymes, peptides, antisense oligonucleotides and RNA interference (RNAi) triggers that are too large to enter cells by themselves. So success here requires the development of safe and efficient means to deliver macromolecules into cells in preclinical animal models and in clinical trials. The use of arginine-rich cationic PTDs/CPPs has grown tremendously since their first appearance in the 1990s [13-16], and the multitude of different molecules that are now being delivered by these peptides in vivo, is continuously expanding. The details of the plethora of cargos, formulations, targets and model systems investigated is beyond the scope of this single review, but notably it includes such diverse compounds as transcription factors [56-58], enzymes [16,59,60], peptides [61-64], proteins [13,14,65-68] and oligonucleotides [54,69-72]. Collectively, these approaches have been used to alter various biological responses in many different ways, including modulation of transcription, apoptosis, inflammation, autophagy, proliferation, differentiation or restoration of protein functions. Among the different cargos that have been delivered, siRNAs and other regulatory nucleotide-based molecules have over the last decade surged as highly desirable therapeutic prospects. The fact that siRNAs can be designed to target any mRNA and that one delivered siRNA has potential to degrade high number of mRNAs when incorporated into the Argonaut-complex make them top-of-the-line drug candidates. However, the large size and negative charge of RNA oligonucleotides make them very difficult to deliver across the cell membrane. Thus, significant attention has been directed on finding ways to get exogenously synthesized RNA molecules into cells in vivo. There are today many different studies and approaches to transduce siRNAs using PTDs/CPPs, ranging from non-covalent complex formations to covalent conjugations of various kinds that can help guide siRNAs into cells [73]. Our group has also contributed with two TATPTD-based delivery strategies for siRNA therapeutics. First, by utilizing a double-strand RNA-binding domain coupled

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to TAT-PTDs [69]. This fusion protein binds to and ‘masks’ the siRNA charge allowing for TAT-PTD-mediated delivery in vitro, and in vivo. More recently, we developed short interfering RiboNucleic Neutral (siRNN) prodrugs [70]. siRNNs are synthesized with bioreversible phosphotriester groups that directly neutralize the phosphate charge on RNAs. Once inside the cells, thioesterases enzymatically cleave off the modifications, leading to the release of a native, charged siRNA that induces RNAi responses. TAT-PTD and other targeting peptides, such as GalNac or RGD, are directly conjugated onto the siRNNs phosphotriester groups. Another exciting area that has expanded in recent years is the design and use of activatable PTDs/CPPs. Activatable PTDs/CPPs are designed so that they are dependent on an initial activation step by proteases or reductases that ultimately leads to release of a functional PTD/CPP. By utilizing, site-, tissue- or tumor-specific enzymes, this approach provides elegant ways of targeting the distribution and uptake to certain sites and tissues with potential to enhance the delivery at desirable areas while lowering possible systemic side effects. Jiang et al. first designed a mechanism in 2004, where a negatively charged PTD/CPP-blocking domain could be cleaved away by proteases [74]. They utilized this approach to specifically image tumors that had high amount of matrix metalloproteases. Similar designs have subsequently been published utilizing matrix metalloproteases, caspases or other enzymes [75]. Activatable PTDs/CPPs coupled to fluorescent probes have potential to assist as molecular navigation tools during surgery [76,77]. Furthermore, when combined to classical targeting peptides, such as RGD or GalNac, they can become a very useful addition to enhance specific uptake at precise target sites. An important aspect of the usage and application of PTDs/ CPPs is toxicity. The balance of the peptide design in terms of size, charge and residue composition will influence both uptake efficiency as well as cytotoxicity. Having too few charged residues will hamper cell association and uptake [26]. Having too long cationic polymers on the other hand is known to bind very strong to cells and may cause membrane ruptures and cell stress. TAT-PTD and 8R alone display only very mild cytotoxic effects even up to ~ 50 -- 100 µM concentration in vitro, [78,79]. Penetratin/Antp have been associated with a slightly higher toxicity profile and other, more amphipathic, peptides show even higher toxic effects [78,79]. Comprehensive comparative toxicity and immunogenicity studies of various PTDs/CPPs in in vivo models are still sorely lacking [79]. However, there are a couple of studies that have investigated this topic. Suhorutsenko et al. tested a handful of PTDs/CPPs, including TAT-PTD, by intravenous (i.v.) injections into mice and assayed for levels of IL-1b and TNF-a [80]. They did not detect any significant immunogenic responses at the highest tested concentration, 5 mg/kg. Furthermore, intraperitoneal injections of 20 nmol of TATPKC over 14 days in rats did not initiate any detectable toxic effects [81]. The same conclusion was made by Toro et al. 6

when injecting mice with a TAT-PNP molecule [79,59,60]. In contrast, i.v. injections of 5 µmol/kg of 9R peptide conjugated to Cy5 was reported to induce toxicity [79,82]. Collectively, the arginine-rich PTDs/CPPs appear to display an encouragingly low toxicity and immunogenicity response. However, more studies will have to be performed and it is important to remember that attachment of different cargos or functional domains to any PTD/CPP can dramatically affect both the uptake and toxicity profile [78,79]. However, examining the PTD/CPP alone is likely not sufficient to draw universal conclusions. Indeed, it might very well be that differences in cargo account for some of the discrepancies observed between labs discussed above. Thus, optimized, carful toxicity studies will have to be performed and compared for each cargo, targeting domain, or endosomal escape motif that are attached to the PTD/CPP. There has been a recent increase in peptide-based drugs [83], and PTD/CPP-based delivery strategies have made it to the clinical trial stage. However, the real surge in using these delivery peptides as drugs is still waiting to happen. By our count, there have been > 25 clinical trials utilizing cationic PTDs/CPPs, predominantly with TAT-PTD, 8R and Penetratin/Antp [7]. Importantly, none of these clinical trials has reported any large adverse effects of PTD-mediated delivery, suggesting that at least at this early date, cationic PTD/ CPP-mediated delivery appears safe for patients. Although there is yet no PTD/CPP-delivered drug that has reached the market, recently a topically applied TAT-PTD formulation, RT001 (Reverence Therapeutics, Newark, California), showed the first statistically significant Phase II clinical trial (p < 0.0001) [17] and is currently being used for an ongoing Phase III clinical trial. ACT1 (FirstString Research) is another promising topically applied peptide that recently showed promising results in a Phase II trial [84,85]. It is not surprising that topical and localized treatment methods provide higher possibility for success at this early age of CPPs/PTDs clinical trials. Local administration facilitates delivery to the target tissue and circumvents some of the obvious obstacles concerning distribution, body clearance and degradation. As new non-cytotoxic endosomal escape and cell-targeting approaches advance from preclinical models, it will be interesting to see if the types of cargo and delivery methods used in clinical trials further broadens and diversifies into entirely new classes of macromolecular therapeutics. Over the next several years, the clinical data should begin to fill in where PTD delivery of macromolecular therapeutics works well and where it does not. 4.

Expert opinion

PTD/CPP-mediated delivery of macromolecular therapeutics has come a long way since the discovery in 1988 that TAT could transduce into cells to transactivate transcription in the nucleus [10-12]. The multitude of experiments and clinical trials that have been performed since then have together shed light on the feasibility and efficacy of PTD/CPP-mediated

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Cationic PTD/CPP-mediated macromolecular delivery: charging into the cell

cellular uptake when linked to the right macromolecular cargo. However, while an impressive amount of ground has been covered, large caveats still remain in our understanding of PTD/CPP transduction. Future studies will need to address important remaining questions involving how to further outline possible toxic effects, incorporate more efficient targetcell/tissue specificity, innovate new peptide designs and improve uptake and endosomal escape efficiencies. All of these topics are of paramount importance for the field and will continue paving the way for new PTD/CPP approaches into clinical trials. The subgroup of arginine-rich PTDs/CPPs delivery peptides have displayed the most promising in vitro and in vivo results so far [1,4,7,16,18-26]. Not surprisingly, these are the best characterized PTDs/CPPs in terms of their molecular functions and interactions with cells. Sophisticated microscopic, inhibitor and phenotypic-based experiments have provided important data regarding uptake, kinetics and how these molecules enter cells by endocytosis [18,21,24-26,30,40-45]. Still, the exact molecular details of how and when argininerich PTDs/CPPs facilitate escape across the endosomal lipid bilayer into the cytoplasm remain shrouded in mystery. Based on phenotypic studies in our lab, delivering siRNAs and Cre recombinase, we guesstimate that < 1% of the PTD/CPPcargo escapes from endosomes into the cytoplasm and nucleus of cells. New and improved uptake assays that can better and more easily distinguish how much of the payload that actually enters the cells and how much that remains stuck nonproductively on the cell membrane or in endosome vesicles would be of tremendous use. In our opinion, it is at least clear that endosomal escape is the rate-limiting step for PTD/CPPmediated delivery and represents an area with huge potential for the field. Our belief is that the next generation of PTD/ CPP delivery agents will undoubtedly have to incorporate novel motifs that dramatically enhance endosomal escape in a non-cytotoxic manner. This is where the real reward for effort put forth will come from. While the enveloped viruses give us a clear sense of how it can be done by membrane destabilization [49,50], incorporating large endosomolytic peptides into PTD/CPP delivery domains will likely be fraught with adverse toxic and/or immune responses that ultimately hinder or dampen such approaches. Therefore, we believe that new smaller peptide or synthetic enhanced endosomal escape domains will have to be developed. Such enhanced endosomal escape domains would have to insert into endosomal membranes in a strictly low pH-dependent manner or take advantage of other endosome-specific features. To achieve this, one may have to use novel chemically modified lysosomotrophic agents, unnatural amino acids or other chemical groups. The key to success for such domains will be to avoid cell membrane-based cytotoxicity while still exerting strong effects inside endosomes.

Because of the small fraction of peptides that actually cross the endosomal membrane into the cytoplasm, it is fair to state that the best transduction results so far have often been obtained with macromolecules that are able to amplify their signals or effects once they enter the cell. In other words, macromolecules that generate high product numbers, such cargos include siRNAs, splice correction oligonucleotides, enzymes and transcription factors [1-3,56-60,52,69-72]. Consequently, for such cargos, it would only take relatively few macromolecules inside the cell to elicit a strong phenotypic response. However, as the efficiency of PTDs/CPPs continues to improve, it may open up opportunities for other, non-enzymatic or amplifying cargos. An additional area where we predict advancements in the near future is within target-cell/tissue specificity. So far, localized application of PTD/CPP therapeutics has advanced the furthest into clinical trials. However, the potential use of tumor/tissue homing peptides, such as RGD, GalNAc and/or other cell surface binding domains [36], gives hope for more specific systemic delivery approaches. Also activatable PTDs/CPPs can potentially help to provide increased specificity. In summary, the optimal arginine-rich PTD/CPP will likely be a design that manages a combination of charge to peptide length with additions of smarter and better endosomal escape features that together favors high uptake with the lowest possible toxicity. The ideal charge is likely going to be close to what the well-utilized TAT-PTD or 8R peptides have. The perfect endosomal escape strategies have still not been elucidated and will be an exciting thing to keep track of the future publications. Enzymes, transcription factors and siRNAs, with amplification of signal capabilities are the preferable cargos, but as the uptake improves we may see better effects for cargos such as peptides, proteins and chemical compounds. Regarding delivery strategies, topical and local treatments will likely continue to be an important approache to utilize PTDs/CPPs, but as the field progresses, we will surely see systemic delivery of combinations of PTDs/CPPs with specific targeting and homing domains. Overall, the advancements in understanding the mechanisms of PTD/ CPP delivery and how to improve it look very encouraging for developing PTD/CPP-based macromolecular therapeutics with potency and specificity exceeding current small molecular drugs.

Declaration of interest The authors were supported by the WM Keck Foundation and the Swedish Research Council. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Expert Opin. Drug Deliv. (2015) 12(10)

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Affiliation

Peter L€onn1,2 & Steven F Dowdy†1 † Author for correspondence 1 UCSD School of Medicine, Department of Cellular and Molecular Medicine, 9500 Gilman Dr., La Jolla, CA 92093-0686, USA E-mail: [email protected] 2 Uppsala University, Science for Life Laboratory, Department of Immunology, Genetics and Pathology, SE-751 08 Uppsala, Sweden

CPP-mediated macromolecular delivery: charging into the cell.

Macromolecular therapeutics, including enzymes, transcription factors, siRNAs, peptides and large synthetic molecules, can potentially be used to trea...
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