- Email: [email protected]
Do Cell-Penetrating Peptides Actually “Penetrate” Cellular Membranes?
© The American Society of Gene & Cell Therapy
mRNA-encoded protein vs. the high-dose yet short-lived recombinant protein? Is it a general distinction between administered recombinant protein and mRNAexpressed product, or is it unique to EPO? DNA plasmids and viral vectors can elicit nonspecific immune responses with detrimental consequences11 and can potentially integrate into the host genome. The mRNA approach is clearly more efficacious than protein administration from the standpoint of sustained production of biologically active products. How it compares to plasmid DNA or viral vectors is not clear, but it might be a moot issue given the considerations mentioned above. mRNA’s safety profile is superior to that of protein, plasmid DNA, or viral vectors. It does not integrate into the host genome during its transient presence; the chemically modified HPLC-purified formulation is nonimmunogenic and does not appear to stimulate neutralizing antibodies. No less important, clinical-grade mRNA can be generated by a relatively cost-effective process—perhaps not as cost-effective as for plasmid DNA but much less costly than production of viral vectors or protein products. Notwithstanding these attractive features, the mRNA approach will be less useful for applications that require extended or lifelong expression of the therapeutic product. The choice of vector will ultimately be determined by weighing the pros and cons of the transient mRNA approach vs. stable gene transfer methods using viral vectors. It will be important to extend these observations to other clinically relevant models in mice (e.g., immunotherapy, clotting deficiencies and surfactant protein-B deficiency) and to confirm the observations in the nonhuman primate model. It is safe to assume that the bar will be much higher in human patients than in animal models, from the standpoints of both effectiveness and safety. Arguably, additional improvements of efficacy, safety, and feasibility will probably be necessary. The primary role of animal modeling is to screen among various options to choose those more likely to perform better in human patients, not to inform whether a particular treatment will exhibit a therapeutic impact in patients and of what magnitude. The report by Karikó and colleagues has helped (me) Molecular Therapy vol. 20 no. 4 april 2012
commentary make a choice, for it is indeed hard to be left unimpressed. Unthinkable a mere decade ago, the pioneering studies from the laboratories of Weissman, Sahin, and a few other investigators have brought closer to reality the concept of using RNA administered directly to patients to express therapeutic proteins and thus even to leapfrog DNA-based approaches for certain applications. References 1.
2.
3.
4.
Karikó, K, Muramatsu, H, Keller, JM and Weissman, D (2012). Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin. Mol Ther, in press. Conry, RM, LoBuglio, AF, Wright, M, Sumerel, L, Pike, MJ, Johanning, F et al. (1995). Characterization of a messenger RNA polynucleotide vaccine vector. Cancer Res 55: 1397–1400. Granstein, RD, Ding, W and Ozawa, H (2000). Induction of anti-tumor immunity with epidermal cells pulsed with tumor-derived RNA or intradermal administration of RNA. J Invest Dermatol 114: 632–636. Kreiter, S, Diken, M, Selmi, A, Tureci, O and Sahin, U (2011). Tumor vaccination using messenger RNA:
prospects of a future therapy. Curr Opin Immunol 23: 399–406. 5. Karikó, K, Ni, H, Capodici, J, Lamphier, M and Weissman, D (2004). mRNA is an endogenous ligand for Toll-like receptor 3. J Biol Chem 279: 12542–12550. 6. Diebold, SS, Kaisho, T, Hemmi, H, Akira, S and Reis e Sousa, C (2004). Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303: 1529–1531. 7. Karikó, K, Muramatsu, H, Welsh, FA, Ludwig, J, Kato, H, Akira, S et al. (2008). Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther 16: 1833–1840. 8. Kormann, MS, Hasenpusch, G, Aneja, MK, Nica, G, Flemmer, AW, Herber-Jonat, S et al. (2011). Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat Biotechnol 29: 154–157. 9. Karikó, K, Muramatsu, H, Ludwig, J and Weissman, D (2011). Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, proteinencoding mRNA. Nucleic Acids Res 39: e142. 10. Casadevall, N, Nataf, J, Viron, B, Kolta, A, Kiladjian, JJ, Martin-Dupont, P et al. (2002). Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin. N Engl J Med 346: 469–475. 11. Takeda, K, Kaisho, T and Akira, S (2003). Toll-like receptors. Annu Rev Immunol 21: 335–376.
Do Cell-Penetrating Peptides Actually “Penetrate” Cellular Membranes? Caroline Palm-Apergi1, Peter Lönn1 and Steven F Dowdy1 doi:10.1038/mt.2012.40
C
ell-penetrating peptides (CPPs), also referred to as peptide transduction domains (PTDs), are polypeptide domains that can enter many, if not most, cell types. CPP/PTDs mediate transduction into cells of a wide range of cargos that otherwise lack bioavailability, such as peptides, proteins, antisense oligonucleotides, and small interfering RNAs. CPP/PTDs are thus being studied extensively as delivery agents for molecular therapies, and a
1 Howard Hughes Medical Institute and Department of Cellular and Molecular Medicine, University of California, San Diego School of Medicine, La Jolla, California, USA Correspondence: Steven F Dowdy, Howard Hughes Medical Institute, Department of Cellular and Molecular Medicine, UCSD School of Medicine, 9500 Gilman Drive, La Jolla, California 92093, USA. E-mail: [email protected]
variety of transducing peptides have now been identified, including both naturally occurring domains and synthetically derived sequences comprising polycationic or amphipathic residues.1 Although much progress has been made in understanding the endocytotic mechanisms by which CPP/PTDs enter cells,1 several important unanswered questions remain. In an upcoming issue of Molecular Therapy, Hirose and colleagues report a study in which they reexamined the direct membrane transduction mechanism of CPP/ PTDs using highly sensitive imaging techniques and well-controlled experimental systems.2 The study reinforces the notion that CPP/PTDs enter cells by endocytosis (macropinocytosis), but it also highlights the significance of the specific peptide and cargo conditions that drive membrane deformations required for concomitant, low-level nonendocytotic uptake. 695
© The American Society of Gene & Cell Therapy
commentary Almost 25 years ago, it was discovered that the transactivator of transcription (TAT) protein from the HIV virus, as well as a chemically synthesized version of TAT, could directly enter cells.3,4 Some years later it was found that a short arginine-rich sequence of TAT was sufficient to deliver or “transduce” macromolecular cargo into cells.5,6 In 1991, Prochiantz and colleagues found that a polypeptide from Antennapedia protein, a homeotic transcription factor from Drosophila, could also transduce into cells.7 A key question that arose from these findings the mechanism for this protein transduction activity. In the 1990s, most CPP/PTDs were presumed to enter cells by directly crossing the cellular membrane in an energy- and temperature-independent manner.8 In 2003, however, Lundberg et al. blew this notion apart by showing that the experimental basis for this proposition was a redistribution artifact caused by fixation before microscopy.9 This led to a rush of studies reexamining how CPP/PTDs enter cells. Although such studies showed that CPP/PTDs enter cells by some form of endocytosis, our laboratory first reported in 2004 that TAT enters cells by macropinocytosis, a specialized form of fluidphase endocytosis that occurs in all cells10 (Figure 1). Futaki’s laboratory reported that same year that octa-arginine (8R) also enters cells by macropinocytosis.11 In our hands, >95% of TAT plus peptide or protein cargos enter cells by macropinocytosis.12 However, there remain ~5% that either is within the error bar of the analysis or perhaps is an indication of a direct cell membrane transduction mechanism that has yet to be resolved. In their new study, Hirose et al. propose that direct penetration of CPP/ PTDs into cells does indeed exist but that (i) it requires a hydrophobic moiety (cargo) attached to the CPP/PTD and (ii) it occurs only at specific locations on the plasma membrane that are competent to induce multivesicular structures along with topical inversions. The investigators evaluated the effect of a somewhat long 12-arginine peptide (R12, as compared with 6 Arg in TAT or 8 Arg in R8) coupled to a hydrophobic Alexa488 fluorophore (R12-Alexa488) on the plasma membrane of HeLa cells.2 Utilizing confocal laser scanning 696
Figure 1 Comparison of cell-penetrating peptide–uptake mechanisms. Transactivator of transcription (TAT) binds to the plasma membrane and enters the cells via macropinocytosis (left), followed by release into the cytoplasm. R12 (12 arginine residues) peptides with hydrophobic cargos directly penetrate the membrane (right) by inducing membrane deformations and multilamellar, particle-like structures at the site of entry.
microscopy and electron microscopy, the authors concluded that R12-Alexa488 enters cells by direct penetration at specific sites where small particle-like cell surface structures are formed. These structures appear to protrude out from the plasma membrane, resembling to some extent a membrane bleb.13 Indeed, the surrounding membrane shows signs of deformation and stains positive for annexin V, a specific phosphatidylserinebinding partner, suggesting some plasma membrane inversion at these R12-Alexa488-induced structures. Surprisingly, no leakage of lactate dehydrogenase from the cells was observed, indicating that the cells remain intact despite the severe membrane perturbations. Furthermore, LAMP-2, a marker of the membrane repair response,14 was detected on the plasma membrane in close proximity to these R12-Alexa488-induced structures. In a startling observation through electron microscopy, the membrane structures were found to contain complex multilamellar lipid membranes (essentially layers of membranes) with internal hollow spaces, suggesting that the R12-Alexa488 peptide initiates gross membrane alterations. Time-lapse microscopy further showed that these structures form within minutes after addition of the R12-Alexa488 CPP/PTD. The R12-Alexa488-containing
vesicles form a large punctuate pattern at the plasma membrane, followed by escape of the R12-Alexa488 into a more even distribution throughout the cytoplasm. Intriguingly, only R12-Alexa488 and R12–hemagglutinin tag were able to initiate the cell surface structures, whereas incubation with very high concentrations (100 mM) of R12 peptide alone did not— nor did a shorter arginine-rich control peptide, R4-Alexa488, suggesting that both peptide cationic arginine length and hydrophobicity of the small cargo are important for this direct-uptake mechanism. This highlights important considerations that must be taken into account with the use of CPP/PTDs conjugated to fluorophores and therapeutically relevant cargo. Both Futaki’s group and Brock’s group15–17 have previously reported the direct membrane transduction of CPP/ PTDs; however, what is unique here is (i) the requirement for a longer arginine-containing CPP/PTD coupled to a small hydrophobic cargo (Alexa488 or hemagglutinin tag) and (ii) the microscopy showing the presence of multilamellar lipid membranes structures at the site of entrance. However, these observations raise more questions than they resolve. First, similar to the studies here, over the years the vast majority of CPP/PTDuptake studies looking at mechanism www.moleculartherapy.org vol. 20 no. 4 april 2012
© The American Society of Gene & Cell Therapy
have relied on Alexa488 or fluorescein isothiocyanate dye–labeled CPP/PTDs and tended to lack phenotypic analysis of cargo function inside of the cells. Given the results here (and, of course, barring all studies that used fixation techniques), we need to question whether these prior studies may have been biased by the particular combinations of CPP/PTD and hydrophobic dye. Second, not all cells in a given population are susceptible to this type of direct-uptake mechanism. By contrast, cellular uptake of CPP/PTDs by macropinocytosis (endocytosis) results in transduction into the entire population of cells, with essentially the same amount of material present inside each cell. Does this suggest an unknown epigenetic contribution or a specific phase of the cell cycle or a definable amount of metabolism (cell growth)? Finally, is there evidence that it occurs in vivo in preclinical models (or in human clinical trials)? Although these will be very difficult experiments to design and control for, they will ultimately tell us whether the cell culture studies are directly related to how these molecules transduce into cells in preclinical animal models as well as in the more than 25 clinical trials using the TAT CPP/PTD. In summary, the study by Hirose and colleagues brings new and useful information to the CPP/PTD field that illustrates the importance of confirming that the peptide and not the cargo is responsible for the observed mechanism(s) of cellular uptake. Although endocytosis may be responsible for the vast majority of CPP/PTD internalization, accumulating evidence suggests that direct penetration does occur at threshold concentrations. In conclusion, the influence of the cargo must be considered when comparing endocytosis and direct penetration, as the present study highlights, and could explain some of the discrepancies that have existed within the field of CPP/PTD uptake for well over 20 years. References
1. Van den Berg, A and Dowdy, SF (2011). Protein transduction domain delivery of therapeutic macromolecules. Curr Opin Biotech 22: 888–893. 2. Hirose, H, Takeuchi, T, Osakada, H, Pujals, S, Katayama, S, Nakase, I et al. (2012). Transient focal membrane deformation induced by arginine-rich peptides leads to their direct penetration into cells. Mol Ther, in press. 3. Frankel, AD and Pabo, CO (1988). Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55: 1189–1193. 4. Green, M and Loewenstein, PM (1988). Autonomous functional domains of chemically synthesized human
Molecular Therapy vol. 20 no. 4 april 2012
commentary
5.
6.
7.
8.
9. 10.
11.
immunodeficiency virus tat trans-activator protein. Cell 55: 1179–1188. Ezhevsky, SA, Nagahara, H, Vocero-Akbani, AM, Gius, DR, Wei, MC and Dowdy, SF (1997). Hypophosphorylation of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in active pRb. Proc Natl Acad Sci 94: 10699–10704. Vives, E, Brodin, P and Lebleu, BA (1997). Truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem 272: 16010–16017. Joliot, A, Pernelle, C, Deagostini-Bazin, H and Prochiantz, A (1991). Antennapedia homeobox peptide regulates neural morphogenesis. Proc Natl Acad Sci USA 88: 1864–1868. Derossi, D, Joliot, AH, Chassaing, G and Prochiantz, A (1994). The third helix of the Antennapedia homeodomain translocates through biological membranes. J Biol Chem 269: 10444–10450. Lundberg, M, Wikström, S and Johansson, M (2003). Cell surface adherence and endocytosis of protein transduction domains. Mol Ther 8: 143–150. Wadia, JS, Stan, RV and Dowdy, SF (2004). Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med 10: 310–315. Nakase, I, Niwa, M, Takeuchi, T, Sonomura, K, Kawabata, N, Koike, Y et al. (2004). Cellular uptake
12. 13. 14.
15.
16.
17.
of arginine-rich peptides: roles for macropinocytosis and actin rearrangement. Mol Ther 10: 1011–1122. Kaplan, IM, Wadia, JS and Dowdy, SF (2005). Cationic TAT peptide transduction domain enters cells by macropinocytosis. J Control Release 102: 247–253. Charras, G and Paluch, E (2008). Blebs lead the way: how to migrate without lamellipodia. Nat Rev Mol Cell Biol 9: 730–736. Palm-Apergi, C, Lorents, A, Padari, K, Pooga, M and Hällbrink, M (2009). The membrane repair response masks membrane disturbances caused by cell-penetrating peptide uptake. FASEB J 23: 214–223. Fretz, MM, Penning, NA, Al-Taei, S, Futaki, S, Takeuchi, T, Nakase, I et al. (2007). Temperature, concentration- and cholesterol-dependent translocation of l- and d-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells. Biochem J 403: 335–342. Kosuge, M, Takeuchi, T, Nakase, I, Jones, AT and Futaki, S (2008). Cellular Internalization and distribution of arginine-rich peptides as a function of extracellular peptide concentration, serum, and plasma membrane associated proteoglycans. Bioconjug Chem 19: 656–664. Duchardt, F, Fotin-Mleczek, M, Schwarz, H, Fischer, R and Brock, R (2007). A comprehensive model for the cellular uptake of cationic cell-penetrating peptides. Traffic 8: 848–866.
Progress in the Development of Hepatitis C Virus Vaccines Hildegund CJ Ertl1 doi:10.1038/mt.2012.30
V
accines save millions of lives each year. Although vaccines are available for many of the viral infections that can be readily prevented by neutralizing antibodies, vaccines for more complex pathogens—including viruses that mutate very rapidly and may require induction of broadly cross-reactive cellular immune responses—remain elusive. Two recent articles1,2 report on vaccine vectors derived from adenoviruses (Ads) of three different species isolated from chimpanzee feces. Vectors encoding antigens from expression cassettes placed into the deleted E1 domain were found to be highly immuno genic in mice and monkeys.1 Even more
1 The Wistar Institute, Philadelphia, Pennsylvania, USA Correspondence: Hildegund CJ Ertl, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. E-mail: [email protected]
important—because mice can lie and monkeys can exaggerate—vectors expressing the NS3-5B region of hepatitis C virus (HCV) genotype 1B induced potent and sustained transgene product–specific CD8+ T-cell responses in human volunteers.2 Traditional vaccines are based on inactivated or attenuated pathogens, purified proteins, or modified toxins. Cellular immunity, especially CD8+ T-cell responses, can best be achieved by gene transfer vehicles that induce de novo synthesis of the vaccine antigens, which are in part cleaved by the proteasome in the cytoplasm. Peptides derived from the degraded antigens are actively transported into the endoplasmic reticulum, where they associate with major histocompatibility class I antigen, and then undergo translocation to the cell surface, where they can interact with the T-cell receptors on CD8+ cells. More than 15 years ago, replicationdefective human serotype 5 adenovirus (AdHu5) vectors originally developed by 697
mRNA-encoded protein vs. the high-dose yet short-lived recombinant protein? Is it a general distinction between administered recombinant protein and mRNAexpressed product, or is it unique to EPO? DNA plasmids and viral vectors can elicit nonspecific immune responses with detrimental consequences11 and can potentially integrate into the host genome. The mRNA approach is clearly more efficacious than protein administration from the standpoint of sustained production of biologically active products. How it compares to plasmid DNA or viral vectors is not clear, but it might be a moot issue given the considerations mentioned above. mRNA’s safety profile is superior to that of protein, plasmid DNA, or viral vectors. It does not integrate into the host genome during its transient presence; the chemically modified HPLC-purified formulation is nonimmunogenic and does not appear to stimulate neutralizing antibodies. No less important, clinical-grade mRNA can be generated by a relatively cost-effective process—perhaps not as cost-effective as for plasmid DNA but much less costly than production of viral vectors or protein products. Notwithstanding these attractive features, the mRNA approach will be less useful for applications that require extended or lifelong expression of the therapeutic product. The choice of vector will ultimately be determined by weighing the pros and cons of the transient mRNA approach vs. stable gene transfer methods using viral vectors. It will be important to extend these observations to other clinically relevant models in mice (e.g., immunotherapy, clotting deficiencies and surfactant protein-B deficiency) and to confirm the observations in the nonhuman primate model. It is safe to assume that the bar will be much higher in human patients than in animal models, from the standpoints of both effectiveness and safety. Arguably, additional improvements of efficacy, safety, and feasibility will probably be necessary. The primary role of animal modeling is to screen among various options to choose those more likely to perform better in human patients, not to inform whether a particular treatment will exhibit a therapeutic impact in patients and of what magnitude. The report by Karikó and colleagues has helped (me) Molecular Therapy vol. 20 no. 4 april 2012
commentary make a choice, for it is indeed hard to be left unimpressed. Unthinkable a mere decade ago, the pioneering studies from the laboratories of Weissman, Sahin, and a few other investigators have brought closer to reality the concept of using RNA administered directly to patients to express therapeutic proteins and thus even to leapfrog DNA-based approaches for certain applications. References 1.
2.
3.
4.
Karikó, K, Muramatsu, H, Keller, JM and Weissman, D (2012). Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin. Mol Ther, in press. Conry, RM, LoBuglio, AF, Wright, M, Sumerel, L, Pike, MJ, Johanning, F et al. (1995). Characterization of a messenger RNA polynucleotide vaccine vector. Cancer Res 55: 1397–1400. Granstein, RD, Ding, W and Ozawa, H (2000). Induction of anti-tumor immunity with epidermal cells pulsed with tumor-derived RNA or intradermal administration of RNA. J Invest Dermatol 114: 632–636. Kreiter, S, Diken, M, Selmi, A, Tureci, O and Sahin, U (2011). Tumor vaccination using messenger RNA:
prospects of a future therapy. Curr Opin Immunol 23: 399–406. 5. Karikó, K, Ni, H, Capodici, J, Lamphier, M and Weissman, D (2004). mRNA is an endogenous ligand for Toll-like receptor 3. J Biol Chem 279: 12542–12550. 6. Diebold, SS, Kaisho, T, Hemmi, H, Akira, S and Reis e Sousa, C (2004). Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303: 1529–1531. 7. Karikó, K, Muramatsu, H, Welsh, FA, Ludwig, J, Kato, H, Akira, S et al. (2008). Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther 16: 1833–1840. 8. Kormann, MS, Hasenpusch, G, Aneja, MK, Nica, G, Flemmer, AW, Herber-Jonat, S et al. (2011). Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat Biotechnol 29: 154–157. 9. Karikó, K, Muramatsu, H, Ludwig, J and Weissman, D (2011). Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, proteinencoding mRNA. Nucleic Acids Res 39: e142. 10. Casadevall, N, Nataf, J, Viron, B, Kolta, A, Kiladjian, JJ, Martin-Dupont, P et al. (2002). Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin. N Engl J Med 346: 469–475. 11. Takeda, K, Kaisho, T and Akira, S (2003). Toll-like receptors. Annu Rev Immunol 21: 335–376.
Do Cell-Penetrating Peptides Actually “Penetrate” Cellular Membranes? Caroline Palm-Apergi1, Peter Lönn1 and Steven F Dowdy1 doi:10.1038/mt.2012.40
C
ell-penetrating peptides (CPPs), also referred to as peptide transduction domains (PTDs), are polypeptide domains that can enter many, if not most, cell types. CPP/PTDs mediate transduction into cells of a wide range of cargos that otherwise lack bioavailability, such as peptides, proteins, antisense oligonucleotides, and small interfering RNAs. CPP/PTDs are thus being studied extensively as delivery agents for molecular therapies, and a
1 Howard Hughes Medical Institute and Department of Cellular and Molecular Medicine, University of California, San Diego School of Medicine, La Jolla, California, USA Correspondence: Steven F Dowdy, Howard Hughes Medical Institute, Department of Cellular and Molecular Medicine, UCSD School of Medicine, 9500 Gilman Drive, La Jolla, California 92093, USA. E-mail: [email protected]
variety of transducing peptides have now been identified, including both naturally occurring domains and synthetically derived sequences comprising polycationic or amphipathic residues.1 Although much progress has been made in understanding the endocytotic mechanisms by which CPP/PTDs enter cells,1 several important unanswered questions remain. In an upcoming issue of Molecular Therapy, Hirose and colleagues report a study in which they reexamined the direct membrane transduction mechanism of CPP/ PTDs using highly sensitive imaging techniques and well-controlled experimental systems.2 The study reinforces the notion that CPP/PTDs enter cells by endocytosis (macropinocytosis), but it also highlights the significance of the specific peptide and cargo conditions that drive membrane deformations required for concomitant, low-level nonendocytotic uptake. 695
© The American Society of Gene & Cell Therapy
commentary Almost 25 years ago, it was discovered that the transactivator of transcription (TAT) protein from the HIV virus, as well as a chemically synthesized version of TAT, could directly enter cells.3,4 Some years later it was found that a short arginine-rich sequence of TAT was sufficient to deliver or “transduce” macromolecular cargo into cells.5,6 In 1991, Prochiantz and colleagues found that a polypeptide from Antennapedia protein, a homeotic transcription factor from Drosophila, could also transduce into cells.7 A key question that arose from these findings the mechanism for this protein transduction activity. In the 1990s, most CPP/PTDs were presumed to enter cells by directly crossing the cellular membrane in an energy- and temperature-independent manner.8 In 2003, however, Lundberg et al. blew this notion apart by showing that the experimental basis for this proposition was a redistribution artifact caused by fixation before microscopy.9 This led to a rush of studies reexamining how CPP/PTDs enter cells. Although such studies showed that CPP/PTDs enter cells by some form of endocytosis, our laboratory first reported in 2004 that TAT enters cells by macropinocytosis, a specialized form of fluidphase endocytosis that occurs in all cells10 (Figure 1). Futaki’s laboratory reported that same year that octa-arginine (8R) also enters cells by macropinocytosis.11 In our hands, >95% of TAT plus peptide or protein cargos enter cells by macropinocytosis.12 However, there remain ~5% that either is within the error bar of the analysis or perhaps is an indication of a direct cell membrane transduction mechanism that has yet to be resolved. In their new study, Hirose et al. propose that direct penetration of CPP/ PTDs into cells does indeed exist but that (i) it requires a hydrophobic moiety (cargo) attached to the CPP/PTD and (ii) it occurs only at specific locations on the plasma membrane that are competent to induce multivesicular structures along with topical inversions. The investigators evaluated the effect of a somewhat long 12-arginine peptide (R12, as compared with 6 Arg in TAT or 8 Arg in R8) coupled to a hydrophobic Alexa488 fluorophore (R12-Alexa488) on the plasma membrane of HeLa cells.2 Utilizing confocal laser scanning 696
Figure 1 Comparison of cell-penetrating peptide–uptake mechanisms. Transactivator of transcription (TAT) binds to the plasma membrane and enters the cells via macropinocytosis (left), followed by release into the cytoplasm. R12 (12 arginine residues) peptides with hydrophobic cargos directly penetrate the membrane (right) by inducing membrane deformations and multilamellar, particle-like structures at the site of entry.
microscopy and electron microscopy, the authors concluded that R12-Alexa488 enters cells by direct penetration at specific sites where small particle-like cell surface structures are formed. These structures appear to protrude out from the plasma membrane, resembling to some extent a membrane bleb.13 Indeed, the surrounding membrane shows signs of deformation and stains positive for annexin V, a specific phosphatidylserinebinding partner, suggesting some plasma membrane inversion at these R12-Alexa488-induced structures. Surprisingly, no leakage of lactate dehydrogenase from the cells was observed, indicating that the cells remain intact despite the severe membrane perturbations. Furthermore, LAMP-2, a marker of the membrane repair response,14 was detected on the plasma membrane in close proximity to these R12-Alexa488-induced structures. In a startling observation through electron microscopy, the membrane structures were found to contain complex multilamellar lipid membranes (essentially layers of membranes) with internal hollow spaces, suggesting that the R12-Alexa488 peptide initiates gross membrane alterations. Time-lapse microscopy further showed that these structures form within minutes after addition of the R12-Alexa488 CPP/PTD. The R12-Alexa488-containing
vesicles form a large punctuate pattern at the plasma membrane, followed by escape of the R12-Alexa488 into a more even distribution throughout the cytoplasm. Intriguingly, only R12-Alexa488 and R12–hemagglutinin tag were able to initiate the cell surface structures, whereas incubation with very high concentrations (100 mM) of R12 peptide alone did not— nor did a shorter arginine-rich control peptide, R4-Alexa488, suggesting that both peptide cationic arginine length and hydrophobicity of the small cargo are important for this direct-uptake mechanism. This highlights important considerations that must be taken into account with the use of CPP/PTDs conjugated to fluorophores and therapeutically relevant cargo. Both Futaki’s group and Brock’s group15–17 have previously reported the direct membrane transduction of CPP/ PTDs; however, what is unique here is (i) the requirement for a longer arginine-containing CPP/PTD coupled to a small hydrophobic cargo (Alexa488 or hemagglutinin tag) and (ii) the microscopy showing the presence of multilamellar lipid membranes structures at the site of entrance. However, these observations raise more questions than they resolve. First, similar to the studies here, over the years the vast majority of CPP/PTDuptake studies looking at mechanism www.moleculartherapy.org vol. 20 no. 4 april 2012
© The American Society of Gene & Cell Therapy
have relied on Alexa488 or fluorescein isothiocyanate dye–labeled CPP/PTDs and tended to lack phenotypic analysis of cargo function inside of the cells. Given the results here (and, of course, barring all studies that used fixation techniques), we need to question whether these prior studies may have been biased by the particular combinations of CPP/PTD and hydrophobic dye. Second, not all cells in a given population are susceptible to this type of direct-uptake mechanism. By contrast, cellular uptake of CPP/PTDs by macropinocytosis (endocytosis) results in transduction into the entire population of cells, with essentially the same amount of material present inside each cell. Does this suggest an unknown epigenetic contribution or a specific phase of the cell cycle or a definable amount of metabolism (cell growth)? Finally, is there evidence that it occurs in vivo in preclinical models (or in human clinical trials)? Although these will be very difficult experiments to design and control for, they will ultimately tell us whether the cell culture studies are directly related to how these molecules transduce into cells in preclinical animal models as well as in the more than 25 clinical trials using the TAT CPP/PTD. In summary, the study by Hirose and colleagues brings new and useful information to the CPP/PTD field that illustrates the importance of confirming that the peptide and not the cargo is responsible for the observed mechanism(s) of cellular uptake. Although endocytosis may be responsible for the vast majority of CPP/PTD internalization, accumulating evidence suggests that direct penetration does occur at threshold concentrations. In conclusion, the influence of the cargo must be considered when comparing endocytosis and direct penetration, as the present study highlights, and could explain some of the discrepancies that have existed within the field of CPP/PTD uptake for well over 20 years. References
1. Van den Berg, A and Dowdy, SF (2011). Protein transduction domain delivery of therapeutic macromolecules. Curr Opin Biotech 22: 888–893. 2. Hirose, H, Takeuchi, T, Osakada, H, Pujals, S, Katayama, S, Nakase, I et al. (2012). Transient focal membrane deformation induced by arginine-rich peptides leads to their direct penetration into cells. Mol Ther, in press. 3. Frankel, AD and Pabo, CO (1988). Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55: 1189–1193. 4. Green, M and Loewenstein, PM (1988). Autonomous functional domains of chemically synthesized human
Molecular Therapy vol. 20 no. 4 april 2012
commentary
5.
6.
7.
8.
9. 10.
11.
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Progress in the Development of Hepatitis C Virus Vaccines Hildegund CJ Ertl1 doi:10.1038/mt.2012.30
V
accines save millions of lives each year. Although vaccines are available for many of the viral infections that can be readily prevented by neutralizing antibodies, vaccines for more complex pathogens—including viruses that mutate very rapidly and may require induction of broadly cross-reactive cellular immune responses—remain elusive. Two recent articles1,2 report on vaccine vectors derived from adenoviruses (Ads) of three different species isolated from chimpanzee feces. Vectors encoding antigens from expression cassettes placed into the deleted E1 domain were found to be highly immuno genic in mice and monkeys.1 Even more
1 The Wistar Institute, Philadelphia, Pennsylvania, USA Correspondence: Hildegund CJ Ertl, The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. E-mail: [email protected]
important—because mice can lie and monkeys can exaggerate—vectors expressing the NS3-5B region of hepatitis C virus (HCV) genotype 1B induced potent and sustained transgene product–specific CD8+ T-cell responses in human volunteers.2 Traditional vaccines are based on inactivated or attenuated pathogens, purified proteins, or modified toxins. Cellular immunity, especially CD8+ T-cell responses, can best be achieved by gene transfer vehicles that induce de novo synthesis of the vaccine antigens, which are in part cleaved by the proteasome in the cytoplasm. Peptides derived from the degraded antigens are actively transported into the endoplasmic reticulum, where they associate with major histocompatibility class I antigen, and then undergo translocation to the cell surface, where they can interact with the T-cell receptors on CD8+ cells. More than 15 years ago, replicationdefective human serotype 5 adenovirus (AdHu5) vectors originally developed by 697