- freely available
Pharmaceuticals 2010, 3(3), 448-470; doi:10.3390/ph3030448
Abstract: Viral diseases affect hundreds of millions of people worldwide, and the few available drugs to treat these diseases often come with limitations. The key obstacle to the development of new antiviral agents is their delivery into infected cells in vivo. Cell-penetrating peptides (CPPs) are short peptides that can cross the cellular lipid bilayer with the remarkable capability to shuttle conjugated cargoes into cells. CPPs have been successfully utilized to enhance the cellular uptake and intracellular trafficking of antiviral molecules, and thereby increase the inhibitory activity of potential antiviral proteins and oligonucleotide analogues, both in cultured cells and in animal models. This review will address the notable findings of these studies, highlighting some promising results and discussing the challenges CPP technology has to overcome for further clinical applications.
1. Nature and Scope of the Challenges Presented by Viral Infections
Viral diseases affect hundreds of millions of people worldwide, resulting in a devastating toll on human health and socio-economic development. Along with the emergence of newly-recognized human pathogens (the SARS coronavirus, the recent influenza viruses H5N1 and H1N1), the ever-increasing incidence of chronic viral infections caused by HIV and hepatitis B and C viruses continues to increase the global burden of infectious diseases [1,2]. Vaccines have been developed for some of the most important viral pathogens. Although vaccines against HIV  and hepatitis C virus  are in clinical phases III and II respectively, there is still little prospect of effective vaccines against these agents. There are enormous challenges to the development of these vaccines, especially since millions of people are already chronically infected with these viruses, which would require therapeutic vaccines for control.
These challenges emphasize the importance of chemotherapy to treat these viral infections . There are around 40 antiviral compounds in clinical use targeting various viral diseases (over half of these drugs are being used in the treatment of patients with HIV infection) while there is no treatment for most acute infections, such as the ones that cause severe illnesses, including hemorrhagic fever, encephalitis, and even cancer [6,7,8,9]. Most of the available drugs are of limited efficacy and come with severe side effects . Importantly, antiviral chemotherapy is plagued by the rapid development of drug resistance strains, resulting from the high rate of replication of viruses combined with the low fidelity with which they replicate their genomes .
In view of the modest existing drug arsenal, the continuing threat posed by viral pathogens urgently calls for the development of novel antiviral agents. As obligate intracellular parasites, viruses present formidable challenges to drug development, the biggest being the in vivo delivery of the antiviral drug into the infected cells, with minimum toxicity to the host cells .
2. The Advent of Cell-Penetrating Peptides
Traditional antiviral therapy has relied on small molecules such as protease inhibitors or nucleotide analogues to inhibit viral enzymes. In the last decades, proteins and nucleic acid molecules have shown very promising antiviral properties. However, due to their physical properties, such as size or hydrophilicity, the cellular uptake of these molecules is strongly restricted. The inability of these molecules to cross the cell membrane to reach their intracellular target precludes further clinical application. The cellular delivery of these molecules has thus arisen as a cornerstone for therapeutic development.
Current techniques for cellular delivery of antivirals include targeted liposomes in cell culture and in mice [11,12,13,14], receptor-mediated endocytosis through antibody binding in cell culture and in mice , retroviral vectors in cell culture , as well as adenoviruses in vaccine delivery . However these techniques present certain limitations and concerns: low efficiency [11,15], immunogenicity [14,15], stability and rapid clearance in bloodstream  for liposomal reagents, and immunogenicity and viral integration on host gene expression for viral vectors [15,19].
A novel strategy to efficiently overcome the impermeable cell barrier came from the surprising findings in the late 1980s that certain naturally occurring short peptide sequences have the ability to enter cells when added to culture media. The tat peptide, derived from HIV-1 transcriptional activator protein (tat) [20,21], and penetratin, derived from Drosophila antennapedia (Antp) transcription protein , were the first cell-penetrating peptides (CPPs) to be described. Tat and penetratin have paved the way to the discovery of other naturally occurring CPPs such as the herpesvirus tegument protein VP22 [23,24] or the cell wall protein-derived peptide inv3 from Mycobacterium tuberculosis [25,26]. Chimaeric CPPs such as transportan (a chimera of the neuropeptide galanin and the wasp venom toxin mastoparan)  and totally synthetic CPPs such as the model amphipathic peptide (MAP)  or arginine oligomers  have also been designed and are routinely used. The exact mechanism of cellular uptake is not clear and studies remain controversial. Current models include uptake through transient pore formation, caveolae, clathrin-dependent endocytosis, and macropinocytosis. Recent data suggest endocytosis as the prevailing model for uptake, although several mechanisms may coexist and differ depending on CPPs [30,31].
What makes these peptides very attractive as a delivery system is their ability to promote intracellular uptake of conjugated cargoes . CPPs have successfully improved the cellular uptake of various cargoes including proteins [26,33], nucleic acids (oligonucleotides [34,35], peptide-nucleic acids [36,37], siRNAs [38,39]), nanoparticules  and liposomes  in a wide range of cells: mainly in mammalian cells, but also in bacteria [37,42], yeasts  and protozoan parasites [43,44]. CPPs have proven to be very efficient in delivering molecules into cells that are refractory to transfection such as primary lymphocytes . In 1999, Schwarze et al. successfully delivered into all tissues in mice the 120-kDa tat-conjugated β-galactosidase protein, which retained its enzymatic activity . Impressively, the conjugate had also crossed the blood-brain barrier and reached the brain tissues, which is usually restricted to small and highly lipophilic peptides. CPPs offer the opportunity to deliver therapeutic molecules that are 200 times larger than the current bioavailability size restriction . CPP-mediated delivery of bioactive compounds into model organisms for cancer , cardiomyopathy , stroke , muscular dystrophy  and viral infections, support the strong therapeutic potential exerted by CPPs. Clinical trials are taking CPP-based drugs a step closer to therapeutic applications. An heptamer arginine-conjugated cyclosporine A (Psorban) has entered phase II of a clinical trial for topical treatment of psoriasis [29,51]. The oligoarginine peptide allows the penetration of cyclosporine A into cells throughout the otherwise impermeable epidermis and dermis.
This review will focus on studies that explored the use of CPPs to deliver antisense agents into virus-infected cells and animal models (Table 1).
|Antiviral cargo||Targeted virus||Conjugated CPP||Experimental systems||Limitations – CPP composition requirements|
|West Nile virus [66,67]||(RXR)4XB||Cell culture||Dose-dependent toxicity in cell culture and mice|
|Japanese encephalitis virus ||(RXR)4XB||Cell culture|
|St. Louis encephalitis virus ||(RXR)4XB||Cell culture|
|Dengue virus [68,69,70]||(RXR)4XB||Cell culture|
|SARS coronavirus ||R9F2C||Cell culture|
|Mouse hepatitis virus [59,63]||R9F2C|
|PPMO toxicity in mice when treatment given after MHV challenge|
|PPMO with higher number of arginine residues exhibit greater antiviral activity in cell culture|
|PPMO with insertions of 6-aminohexanoic acid offer greater protection in mouse|
|Equine arteritis virus ||R9F2C||Cell culture|
|Porcine reproductive and respiratory syndrome virus ||R5F2R4C||Cell culture|
|Poliovirus 1 ||R9F2C||Cell culture||in vitro toxicity when longer periods of treatment|
|Human rhinovirus 14||R9F2C||Cell culture|
|Coxsackievirus B2||R9F2C||Cell culture|
|Coxsackievirus B3||(RXR)4XB||Cell culture|
|Foot-and-mouth disease virus ||R9F2C||Cell culture|
|Sindbis virus ||R9F2C||Cell culture|
|Venezuelan equine encephalitis virus ||(RXR)4XB||Cell culture|
|Ebola virus [64,78]||(RXR)4XB||Cell culture|
|PPMO with insertions of 6-aminohexanoic acid and higher number of arginine-6-aminohexanoic repeats offer greater protection in mouse|
|Respiratory syncytial virus ||(RXR)4XB||Cell culture||Endosomal entrapment|
|Measles virus ||(RXR)4XB||Cell culture|
|Influenza A virus [81,82,83]||(RXR)4XB||Cell culture||Higher doses of PPMO induced abnormal infiltration of mouse lungs by immune system cells|
|Kaposi’s sarcoma-associated herpesvirus [84,85]||R5F2R4C||Cell culture|
|Herpesvirus type 1||(RXR)4XB||Cell culture|
|PNA||HIV-1 [91,92,93,94,95,99,100,101]||Disulfide-linked:||Cell culture|
Some preliminary mouse studies for tat and penetratin conjugates
|Endosomal entrapment requiring lysosomotropic agents|
Nature of CPP and of CPP-PNA linkage had an effect on conjugate activity
|Japanese encephalitis virus ||tat||Cell culture|
|siRNA||Hepatitis C virus ||tat||Cell culture|
|HIV-1 ||nonamer arginine (9R)||Mouse|
|Proteins||HIV-1 [103,104,105]||tat||Cell culture|
|Human papillomavirus type 18 ||9R||Cell culture||Nature of CPP directly impacted the level of antiviral activity|
3. Delivery of Antisense Agents
The past decade has witnessed the breakthrough of gene-silencing antisense agents including antisense oligonucleotides (AOS), AOS analogues, ribozymes, DNAzymes and the popular siRNAs. This novel approach is based on the discovery 30 years ago that nucleic acid molecules could be used to specifically target complementary viral nucleic acid sequences and inhibit viral replication [52,53]. With these new promising molecules comes the ability to simultaneously target multiple viral sequences, which would preclude the selection of drug resistance.
3.1. Delivery of Phosphorodiamidate Morpholino Oligomers (PMOs)
So far, conjugation of CPPs to phosphorodiamidate morpholino oligomers (PMOs) has clearly been the most popular and most promising use of CPPs in antiviral development. PMOs are antisense DNA oligonucleotide analogues . Their backbone is composed of morpholine rings joined by uncharged phosphodiamidate linkages, in place of the sugar and anionic phosphodiester linkage of DNA. They are typically synthesized as 20–25-base long oligomers. They offer great features for clinical applications: they are water-soluble, nuclease-resistant, and their uncharged backbone interacts weakly with serum and cellular proteins thereby reducing toxicity . PMOs are mainly designed to interact with 5’ and 3’ non-coding regions of the target mRNA, AUG-codon translation start-site region or splice junctions. They act through steric blockage of the complementary RNA sequences, precluding proper mRNA processing or translation and thus reducing target protein levels and viral replication (Figure 1).
As PMOs do not efficiently enter cells on their own, investigators have been routinely arming them with CPPs to promote their uptake into virus-infected cells and enhance their antisense efficacy [56,57]. These peptide-conjugated PMOs are referred to as PPMOs in the literature. As highlighted by Moulton and Jiang , PMOs are particularly well-suited cargoes for CPPs as their uncharged components do not interact with the positively-charged carrier peptide thereby not interfering with its membrane binding properties. The first report of PPMO antiviral activity in 2004  has fueled the increasing interest in PMO-technology to inhibit viral infections as witnessed by more than 20 in vitro and/or in vivo studies published on antiviral PPMOs within the last five years (Table 1). Studies on RNA viruses have been extensively reviewed by Stein  with the latest in vivo work discussed by Moulton and Jiang . We will focus here on the overall findings in regards to the cell penetrating moiety of the conjugates, and we will then review the studies on targeting DNA viruses.
3.1.1. Nature of Peptides Conjugated to PMOs
Peptide-mediated PMO delivery has been the focus of intense efforts to identify active, stable, non toxic and endosomolytic CPPs, of which (RXR)4XB (also called P7; R = L-arginine, X = 6-aminohexanoic acid; B = β-alanine) has proven very successful [61,62]. In antiviral studies, PMOs have been covalently linked to various arginine-rich peptides, the most frequent being R9F2C (R = L-arginine; F = L-phenylalanine; C = L-cysteine), R5F2R4C (also called P4), and (RXR)4XB. Conjugation is mainly at the 5’-terminal end of the PMO. In studies comparing the activity of various CPPs against murine hepatitis virus (MHV)  and Ebola virus (EBOV) , arginine-rich peptides with insertions of 6-aminohexanoic acid stood out for their in vivo effectiveness. The higher the number of arginine-6-aminohexanoic repeats, the higher were PMO antiviral effect on MHV in cell culture  and the protection against EBOV infection in mice . These findings agree with the results from Amantana et al. , Abes et al.  and Youngblood et al. , who also found that these residues increased serum stability and enhanced endosomal escape of the conjugates. Burrer et al. documented that the nature of the linker (XB or cysteine) did not affect PPMO in vitro effectiveness against MHV or cytotoxicity, although the insertion of β-alanine residues was reported to improve both serum and intracellular stability of PPMO conjugates .
3.1.2. PPMOs against RNA Viruses
PPMO antiviral activity has mainly been exploited against a wide range of non-retroviral RNA viruses. In cell culture, PPMOs were tested against West Nile virus (WNV) [66,67], Japanese encephalitis virus , St. Louis encephalitis virus , dengue virus (DENV) [68,69,70], severe acute respiratory syndrome coronavirus (SARS-CoV) , MHV [59,63], equine arteritis virus , porcine reproductive and respiratory syndrome virus , foot-and-mouth disease virus , poliovirus 1 (PV1) , human rhinovirus 14 , coxsackievirus B2 , coxsackievirus B3 (CVB3) , Sindbis virus , Venezuelan equine encephalitis virus (VEEV) , EBOV [64,78], respiratory syncytial virus (RSV) , measles virus , and influenza A virus (FLUAV) [81,82].
Cell culture assays consistently showed that PPMO readily entered infected cells and could specifically and strongly inhibit viral replication. Fluorescein-labeled PPMOs accumulated in the nucleus but were present throughout the cells [66,79]. Lai et al.  reported punctuate foci of signal in the cell cytoplasm which might be indicative of endosomal entrapment of PPMO.
Many investigators identified potent PPMO activity in cell culture assays and went on to test their antiviral properties in murine experimental models. Most of the investigators explored both the prophylactic and therapeutic potential of PPMO and the best protection was usually achieved with pre-infection and post-infection PPMO treatments. PPMOs were shown to suppress viral replication, attenuate symptoms of disease in and/or increase survivorship of mice infected with WNV , DENV , MHV , PV1 , CVB3 , VEEV , EBOV [64,78], RSV , and FLUAV [82,83]. Some PPMOs demonstrated very profound and impressive antiviral effects. In particular, two 5-μg pre-infection doses of (RX8)B-conjugated PMOs completely protected mice against lethal challenge with EBOV [64,78]. Mice treated with 200-μg doses of a (RXR)4XB-conjugated PPMO given pre- and post-infection survived VEEV lethal infection . The authors found that over the 28-day period of monitoring, VEEV was undetectable in PPMO-treated mice brain, blood and peripheral tissues.
3.1.3. PPMOs against DNA Viruses
While PPMOs are being routinely tested against RNA viruses, there have only been a handful of studies documenting the use of PPMO technology against DNA viruses. The first report came from Zhang et al. who were interested in blocking Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic replication . They explored the effects of the inhibition of the expression of KSHV latency-associated nuclear antigen (LANA) and replication and transcription activator (RTA), two factors involved in latency maintenance and in the switch to lytic phase, respectively. To do so, the investigators designed PMOs directed against LANA and RTA and covalently conjugated them at their 5’ terminal to R5F2R4C peptide. While fluorescein (Fl)-labeled PMOs entered cells poorly, Fl-PPMOs were efficiently taken up by BCBL-1 lymphocytes which, as emphasized by the authors, are difficult to efficiently transfect with common techniques such as lipofectamine. PPMOs could effectively inhibit RTA and LANA protein expression in cell culture. Treatment of KSHV-infected cells with RTA PPMO suppressed KSHV lytic replication. RTA knock down resulted in reduced levels of proteins expressed downstream of RTA. One of these downstream proteins is viral interleukin-6 (vIL-6) and is thought to be essential for the development of KSVH-associated diseases. In a subsequent study, Zhang et al. blocked the expression of vIL-6 with PPMOs. vIL-6 PMOs were covalently conjugated at their 5’ to (RXR)4XB or R5F2R4C. PPMO treatment inhibited vIL-6 expression, which in turn reduced human IL-6 level and KSHV yield, and reduced growth of treated cells .
Moerdyk-Schauwecker et al. tackled the reactivation of another DNA virus, the herpes simplex virus type 1 (HSV-1) . They investigated the antiviral activities of five (RXR)4XB-conjugated PMOs directed against three HSV-1 immediate-early genes, ICP0, ICP4 and ICP27 crucial for HSV-1 replication. The most potent PPMOs designed against ICP0 or ICP27 strongly inhibited HSV-1 replication in cell culture by reducing viral protein expression (Figure 1). In particular, ICP0 PPMO was able to suppress the replication of several HSV-1 strains, including an acyclovir-resistant strain. The authors also tested ICP0 PPMO in a mouse model of ocular herpes infection. Topical application of 10 μg ICP0 PPMO to the eyes of HSV-1 infected mice inhibited virus replication in mouse eyes and reduced the incidence of eye disease by 37.5–50%, thereby preventing death associated with HSV-1 eye infection. To evaluate in vivo PPMO toxicity, the investigators applied 100 μg ICP0 PPMO (ten times the antiviral dose) daily to the eyes of uninfected mice. No gross or microscopic eye damage, no effect on body weight and temperature and no change of behavior were observed following the seven-day treatment. These results strengthen the potential of PPMOs as a treatment for recurring corneal disease from HSV-1 reactivation.
3.1.4. Enhancement of PMO Antisense Activity
Besides improving PMO cellular uptake, the CPP moiety was also shown to intensify PMO antisense activity against EBOV by 10- to 100-fold in cell-free translation assays . These results are consistent with the findings of Nelson et al. who showed that PPMOs exhibited antisense activity 3- to 25-fold higher than corresponding PMOs while bearing lower off-target effects . The authors propose that the arginine-rich peptides enhance RNA-PMO binding affinity thereby increasing specific antisense activity.
3.1.5. PPMO Toxicity
PPMOs appear to be generally well tolerated by cultured cells and animal recipients. However, some studies reported toxicity in cell culture with increase of PPMO dose  or longer period of exposure [66,75], and in vivo toxicity as the treatment dose increased, usually at doses higher than antiviral ones . The peptide moiety of PPMOs seems to contribute to this toxicity. When Deas et al. tested both PMO and PPMO against WNV in mice, they found that mice could tolerate higher doses of PMO compared to PPMO (3 mg against 300 μg, respectively) . Mice treated with high doses of PPMO would suffer from weight loss and develop abnormal behavior and smaller livers. Besides dose-dependent toxicity, the schedule of treatment regimen may also affect PPMO toxicity: while Burrer et al. did not document any treatment-associated toxicity when PPMOs were administered to healthy mice, they observed significant toxicity when PPMO treatment was given after MHV challenge . Lupfer et al. reported abnormal infiltration of mouse lungs by immune system cells when PPMO targeting FLUAV was administered at higher doses . The authors hypothesized that the arginine-rich CPP mimics the eosinophil major basic protein, also rich in arginine residues, in triggering the migration of inflammatory cells.
3.2. Delivery of Peptide-Nucleic Acids (PNAs)
Peptide-nucleic acids or PNAs are oligonucleotide analogues in which the sugar-phosphate backbone has been replaced with peptide bonds . They are resistant to both nucleases and proteases. They are used as antisense molecules to specifically bind to their complementary DNA or RNA sequences, creating a structural hindrance and inhibiting viral transcription, translation and replication processes.
3.2.2. Preclinical Studies of CPP-PNA Conjugates in Mice
To date, only few in vivo studies have been reported with CPP-PNA conjugates . In the field of antivirals, Pandey and colleagues have started preliminary toxicity, immunological and pharmacokinetic studies in mice for the anti-HIV-1 PNATAR-penetratin conjugate [99,100,101]. Chaubey et al. reported the non-toxicity of the complex when administered at repeat doses ranging up to 100 mg/kg . Mice which were given 100 mg/kg of the conjugate, a dose the authors highlight is far in excess of the expected therapeutic dose, suffered from diarrhea and reduced physical activity and from spleen, liver and kidney serotosis. However they recovered during the 60-day follow-up period with no irreversible organ damage reported.
Upadhyay et al. demonstrated that PNATAR-penetratin conjugate is moderately immunogenic mainly due to its penetratin moiety . Cytokine secretion profiles of the lymph node cells showed elevated levels of proinflammatory cytokines, such as IL-2 and IL-12, which are known to promote proliferation of T lymphocytes and slow down the death of CD4+ T cells. The authors emphasize that these immunogenic properties of the conjugate could prove to be beneficial to the host.
Ganguly et al. studied the tissue distribution and clearance of 125I-labeled PNATAR and its penetratin and tat conjugates . They reported the distribution of the conjugates throughout the mouse major internal organs when administered by oral route as well as their slow release and clearance from different organs. Surprisingly, unconjugated naked PNATAR displayed a similar tissue distribution and clearance profile although the extent of its uptake was lower than its CPP conjugate. In vivo antiviral activity of PNATAR and its penetratin and tat conjugates needs to be assessed.
3.3. Delivery of Small Interfering RNAs (siRNAs)
The use of CPPs to deliver siRNAs into cells has rather been limited to date, even more so for antiviral siRNAs. Järver et al. suggested that siRNAs could be less amenable to CPP delivery due to charge interactions between the peptide and the siRNA and inefficient endosomal escape of the conjugates . To our knowledge, the only report of CPP-mediated delivery of antiviral siRNA is from Meng et al. who constructed a cell-permeable siRNA targeting hepatitis C virus 5’ untranslated region . The siRNA was crosslinked at its 3’ end with the tat peptide. The authors reported the cellular uptake and antiviral effect of the CPP-siRNA as being as efficient as transfection with lipofectamine in Huh-7 cell culture. However, some cell types being refractory to lipofectamine transfection which can moreover be toxic in vivo, CPP-mediated delivery of siRNA is definitely worth investigating in other cell types and in animal models of viral infections.
Although Kumar et al. did not use CPP as a cell delivery system per se, it is noteworthy that they exploited its binding capability to polyanionic nucleic acids such as siRNAs . The authors developed a novel method for systemic delivery of antiviral siRNAs into T cells in mice: they used an antibody to the CD7 receptor as their delivery system by receptor-mediated endocytosis. They bound the siRNA to the antibody through the positively charged nona-D-arginine (9R) peptide conjugated to its C-terminal via a disulfide bond. They successfully suppressed HIV-1 infection in a humanized mouse model using a cocktail of these siRNA complexes, inhibiting HIV-1 entry into target cells and its replication.
The above reviewed studies contain a common message: CPP-conjugated antiviral agents hold great promise as novel preventive and therapeutic modalities against viral infections. The remarkable antiviral effects of PPMOs against lethal infections with EBOV, VEEV, FLUAV and DENV, and against the usually-lethal infections with PV1 and WNV, call for a wider use of CPPs in the delivery of antiviral drugs. CPP-conjugated antivirals also show promise in inhibiting the reactivation from latent infections of viruses such as HSV-1. Moreover, the assessment of CPP antiviral properties and the understanding of their molecular mechanisms could lead to the exploitation of these activities for the development of microbicides to inactivate virions after exposure.
These CPP-antiviral studies illustrate the search for the “perfect” CPP-cargo couple, as CPP carrier efficiency is dependent on the nature of its cargo, affecting the antiviral activity. This is particularly true for PPMO research where much effort has been put towards developing highly active peptides to outperform the more traditionally used penetratin and tat peptides [61,62]. Improving CPP endosomolytical properties to intensify antiviral activity of the conjugated cargo has become a main focus, as the endosomal escape appears to be critical to antiviral activity .
CPP should not only promote the cellular uptake of the cargo but also improve its intracellular trafficking to ensure that it reaches the appropriate cellular compartment. This aspect cannot be overlooked when designing antivirals as different viruses may carry their replication cycles in different subcellular locations such as the cytosol, the nucleus or in an intricate membrane system within the cytosol. The type of linkage between the CPP and the antiviral compound is of importance as for instance a disulphide bond would result in the rapid release of the cargo through the action of cytoplasm glutathione. Therefore the choice of CPP should not only be optimized to the cargo but should also be specific to the virus to ensure that the antiviral molecule efficiently reaches its viral target in the subcellular compartment.
Research on antiviral CPP-PNA, -siRNA and -protein conjugates, although encouraging, is still at its infancy and importantly needs validation in animal models. There is a need for improvement in the design of CPP to tackle the issue of in vivo toxicity and thereby make the drug conjugates safer. As Stein highlights, even though PPMO dose regimens are shown to be nontoxic, it will be necessary to show that treatment with doses several times higher than the expected therapeutic dose is well tolerated . It is noteworthy that all the PPMO studies were done in collaboration with a company specializing in RNA-based drugs, AVI BioPharma Inc, which first developed the PMO and provided the PPMO compounds. Of all the CPP-antiviral investigations, PPMO studies are the ones that are at the most advanced stage, with several reports of use in animal models and directed against a wide range of viruses. The work on PPMO illustrates the successful collaboration between academia and industry.
One recurring challenge in the global control of viral infections is the development of drug-resistant viral strains. It is very likely that escape mutants will arise during treatment, especially if long-term therapy is required. This issue can be overcome if treatment can be easily switched to different antisense molecules. The ease to synthesize a new CPP-PNA, CPP-siRNA or PPMO directed to the mutated sequence needs to be addressed. According to Stein, PPMO production is costly and complex . Most investigators limited their in vivo studies to the most potent PPMO identified in cell culture. However, treatment with a combination of antisense molecules targeting multiple sequences in the viral genome should have a greater antiviral effect compared to a treatment with a single antisense molecule, and should prevent selection of escape variants. More studies with treatment with a cocktail of antivirals would be of critical relevance for further clinical applications.
A number of CPPs are derived from viral proteins, such tat, VP22, and FHV. The role of these cell penetrating motifs during infection has yet to be elucidated but it is interesting to see that another obligate intracellular pathogen, Mycobacterium tuberculosis, also possesses a cell wall protein with such a peptide sequence . CPPs represent an example of how understanding how pathogens interact with human host cells can lead to new ways to treat and prevent viral infections.
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