Steric Block Biological Activities of PeptidePNA Conjugates

The field of peptide-ON conjugates was much stimulated by Corey's early studies that showed a 48 000-fold enhancement of binding to RNA of a synthetic oligodeoxyribonucleotide conjugated to a lysine-rich peptide.71 Soon after, Corey and colleagues described the synthesis of PNA conjugates of the a

(b) a bifunctional PNA used in synthesis of peptide-PNA-peptides.

Penetratin peptide and showed substantially enhanced uptake into DU145 prostate cancer cells compared to that of unconjugated PNA.72 However, much controversy in this field has centred, in particular, around to what extent improvements in cell uptake afforded by an attached peptide are correlated with steric block biological activities of the PNA within cells.

In principle, RNA-targeting applications of PNA may be divided into those that occur in the cytosol, such as inhibition of protein translation, and those that occur in the cell nucleus. A number of RNA targets on an mRNA may be used to block protein translation by PNA, for example the AUG initiator sequence, 5'-terminus and other regions of the 5'-untranslated region (UTR), or even the 3'-UTR, but in general it has not been possible to inhibit translation significantly in cells by PNA targeting the coding region of a gene,73 even though it has been shown that it is possible to block translation in vitro21 Table 4.1 lists a number of PNA-peptides that have been investigated for modulation of gene expression in mammalian cells.

Among the very few model systems studied for protein-expression inhibition by a PNA-peptide, the Langel group, working towards the modification of pain transmission, showed very early on that a 21-mer PNA disulfide-coupled to the CPPs Penetratin or Transportan (a hybrid of a section of the neuro-peptide galanin and the wasp venom peptide mastoparan) blocked expression of the galanin receptor mRNA in human Bowes cells.67 More recently, this group has obtained remarkably potent inhibition (down to 70-80 nM EC50) in the same biological model with shorter PNAs targeting different regions of

Table 4.1 Some recent PNA-peptide conjugates used in gene modulation applications in cells.

PN-peptide conjugate (peptide sequence)

Conjugation type Target/host










(FL)3-PNA Penetratin-PNA

21 -mer



Straight-through synthesis

19-mer (+1 K) Straight-through synthesis 13-mer Disulfide

14-mer (+1 K) Straight-through synthesis

Galanin mRNA/human Bowes cells and rats

Galanin mRNA/human Bowes cells

Nociceptin/orphanin FQ mRNA/neonatal rat cardiomyocytes and CHO cells

Caveolin I mRNA/HeLa and primary endothelial cells RNA component of telomerase/ JR8 cells

Inducible nitric oxide synthase mRNA/RAW 264.7 cells

76 90




Transportan-PNA 18 -mer





R6Penetratin-PNA 18-mer(+4K)


R6Penetratin-PNA 16-mer(+4K)






Luciferase/P-globin aberrant splice site/HeLa pLuc reporter system

Various: Mal-eimide/Ester/ Disulfide

Luciferase/P-globin aberrant splice site/HeLa pLuc reporter system

Straight-through CD40 mRNA/BCL! cells and 85 synthesis primary macrophages

Stable/disulfide Luciferase/P-globin 92

aberrant splice site/HeLa pLuc reporter system

Stable/disulfide TAR RNA/HeLa TAR-Luc re- 58 porter system

Straight-through synthesis

Encapsidation signal duck hepatitis virus/duck primary hepatocytes galanin-receptor mRNA, conjugated to a truncated Transportan version TP10.74 PNA-peptide conjugates have also been used to inhibit telomerase activity, by binding to the RNA component of the enzyme complex. For example, a 13-mer PNA disulfide-linked to Penetratin was able to inhibit tel-omerase activity in JR8 melanoma cells when incubated for 144 hours (IC50 ~ 7 mM).75 PNAs stably linked to short hydrophobic peptides rich in Leu and Phe have shown increased cell internalization and activity in vitro.76 Stimulated macrophages incubated with anti-inducible nitric oxide synthase (iNOS) PNA-peptide showed up to 44% decrease in nitric oxide synthase activity compared to non-targeted controls.

In a different model system, an amphipathic peptide, MAP, disulfide-conjugated to a PNA complementary to the nociceptin-orphanin FQ receptor mRNA was shown by the Bienert research group to impart improved cell uptake and steric block biological effects in both CHO cells and neonatal rat cardiomyocytes.77 A 19-mer PNA complementary to human caveolin 1 mRNA, when linked to a D(AAKK)4 peptide, was able to inhibit expression in HeLa cells (IC50 = 2 mM) and even in primary endothelial cells, but only when added at high (>15 mM) concentrations, whereas the same PNA delivered by liposomal packaging and a complementary DNA sequence had an IC50 of 25 nM.78 A nuclear localization sequence (NLS) and PNA conjugate was completely inactive in this assay.

For inhibition of translation in cells, steric block can rarely match quantitatively the efficacy of RNase H inducing antisense or siRNA, where the RNA is degraded. Most studies, therefore, that involve PNA-peptide conjugates have been directed to nuclear RNA targets, such as the redirection of splicing.4,79 PNA and other steric blocking ONs are particularly well-suited to this application, since siRNA is believed to be active primarily in the cytosol, and conventional PS-containing antisense ONs are generally poorer competitors for the nuclear RNA regions targeted by protein complexes, such as the spliceosome.

Although the cellular uptake pathway of PNA conjugates involving hydro-phobic peptides, such as Transportan, is not yet clear, there is agreement that PNAs conjugated to cationic peptides are taken up primarily by endocytosis into cytosolic vesicles and become sequestered there.39,78,80,81 The key issue for biological activity is thus whether the attached peptide can bring about sufficient release of the PNA from such vesicles, such as can be obtained artificially through co-administration of a lysosomotropic agent (e.g. chloroquine or calcium ions).58,82,83 Peptides that have been classed as 'cell-penetrating' vary substantially in their ability to influence endosomal release and thus allow the PNA to enter the nucleus, as judged by splicing redirection assays.

In a HeLa cell culture assay, the addition of just four lysines on the C-terminus of a PNA 18-mer (PNA-4K) complementary to an aberrant splice site (654) within a DNA construct containing a human p-globin intron within the gene for enhanced green fluorescent protein (EGFP; Figure 4.6) was enough to result in the observation of some splice correction and up-regulation of EGFP.9 The activities of the PNA-4K and PMO oligomers were found to be much higher that those for negatively charged OMe-PS oligomers with free

The EGFP splice-correction model9 in which oligonucleotides or PNA complementary to the 654 site can bring about up-regulation of EGFP. A similar construction has been used with the 705 site to up-regulate luciferase production.87

Figure 4.6

The EGFP splice-correction model9 in which oligonucleotides or PNA complementary to the 654 site can bring about up-regulation of EGFP. A similar construction has been used with the 705 site to up-regulate luciferase production.87

uptake, but nevertheless 3-10 mM concentrations were required to see any significant level of splice correction. However, in transgenic mouse studies with the same EGFP 654 splice-correction model, PNA-4K 654 showed activity in several tissues (heart, kidney, lung, liver, muscles and small intestine) when delivered intraperitoneally (i.p.) at 50mgkg_1 for up to 4 days, and generally showed higher splicing correction (up to 40%) than the corresponding MOE and PMO oligomers.84 No visible signs of pathology were seen for the mice at this dose.

In a different model involving the downregulation of murine CD40 pre-mRNA via splice redirection, it was shown that 8 Lys residues on the N-terminus of a PNA complementary to the splice site increased activity significantly with free uptake, compared to 4 Lys residues, but once again high concentrations of conjugate (3-10 mM) were required to see strong downregulation of this target in either B-cell lymphoma 1 (BCL1) cells or primary murine macrophages.85 Albertshofer et al. examined stably linked PNA conjugates of variously spaced oligo-Lys peptides by free incubation with cells and showed some small enhancements of splicing redirection in certain cases compared to Lys8.86 Preliminary pharmacokinetic studies of Lys8-PNA in mice showed a broad tissue distribution and only modest elimination via excretion within 24 hours.86

The studies were continued with a series of 28 cationic peptides based on Lys or Arg that had amphipathic character.87 This work led to improved peptide-PNA conjugates that showed stronger splicing redirection activity at 1-6 mM concentrations without toxicity, but half-lives of the peptide parts of the conjugates in 25% mouse serum were found to be just a few hours or even less. The PNA itself was found to be stable to nucleases and proteases in serum. Two such peptide-PNA conjugates, in which the peptide part utilized metabolically more stable d-amino acids, were radiolabelled, and studies in mice showed remarkably different biodistributions, demonstrating that peptide sequence alteration may be used to modulate in vivo pharmacology of a PNA. In vivo efficacies for these constructs have not yet been reported, however.

Many of the recent results on the use of CPPs to improve PNA cell delivery have been observed in a splice redirection model developed by the group of Kole.

This model is based on an HeLa cell integrated plasmid test system containing a gene that codes for firefly luciferase but interrupted by an aberrant p-globin intron,88 similar to that already mentioned for EGFP (Figure 4.6),9,84 but where aberrant splicing occurs at a different site (705). Upregulation of luciferase by redirection of splicing is obtained by the introduction into the cells of an 18-mer ON complementary to the aberrant 705 splice site. In a collaboration between our group and that of Bernard Lebleu and colleagues in Montpellier, we found that (Lys)8-PNA stably linked conjugate was not very effective at 1 mM concentration and splicing correction was observed only when the lysosomotropic agent chloroquine was added.83 There was also evidence of some cell cytotoxicity when 2.5 mM or higher concentrations were used. We found also that stably or disulfide-linked conjugates of the Tat (48-60) peptide with the 18-mer PNA had similar sequestration properties in endosomes and that the conjugates were inactive in the splicing assay at 1 mM concentration unless chloroquine was added.89 Thus we concluded that neither (Lys)8 nor Tat constructs are good starting points for CPP-PNA conjugate design.

Bendifallah et al., evaluated a range of peptides, such as Tat, Penetratin, R9 and Transportan, conjugated to a PNA 18-mer in several alternative ways.46 The only conjugates to show significant splicing correction when delivered to the HeLa 705 cells at 2-5 mM were those that contained Transportan, but no simple relationship could be determined between the type or placement of the linker between peptide and PNA and the splicing correction activity, although labile linkages were in general of higher activity. Addition of serum to the test media had a dramatic negative effect on splicing-correction activity in all cases. Very similar results were obtained by El-Andaloussi and colleagues for disulfide-linked conjugates of Tat, Penetratin and Transportan, and only at concentrations of 5-10 mM was significant splice-correction activity seen, and only in the absence of serum.90 Serum suppression of splicing correction activity seems to be a general phenomenon common to most PNA-peptides and PMO-peptides (Chapter 3). Partly, this may result from proteolysis of the peptide part in serum, whereas the PNA is known to be stable, but cationic peptides may also bind to serum proteins.

We have recently evaluated more Arg-rich peptides for conjugation with PNA. Substantial splicing correction was obtained at 2 mM for (R-Ahx-R)4 stably conjugated to PNA 18-mer (where Ahx stands for aminohexanoyl) in the absence of chloroquine,89 at a similar level to that obtained for the (R-Ahx-R)4-Ahx-pAla-PMO construct,91 a lead peptide conjugate for in vivo studies which showed very little in vitro cytotoxicity (see Chapter 3). However, most impressive of all has proved to be a conjugate of the 18-mer PNA disulfide-linked or linked through a stable thioether bridge to an R6-Penetratin peptide. We found that such conjugates gave rise to extremely good luciferase upregulation, corresponding to about 50-60% splicing correction at 1 mM concentration, as judged by a reverse transcription polymerase chain reaction (RT-PCR) assay for transcript length.47 A mutant in which a W residue was replaced by an L residue at position 6 of the Penetratin part of the R6-Penetratin-PNA conjugate showed slightly higher activity, even though such an amino acid substitution had previously been shown to inhibit Penetratin peptide from crossing membranes.92 This suggests that the sequence requirements of the R6-Penetratin peptide needed to impart cell uptake and endosomal release of the PNA are considerably different to those of Penetratin peptide alone. The level of activity seen with Rg-Penetratin-PNA is higher than that for other CPP-PNA constructs published to date. By further peptide-sequence manipulation, hopefully it may be possible to bring the splicing-correction activity levels for PNA-peptides in this model splice-correction system down to the nM range.

PNAs conjugated to peptides have potential also as antiviral or virucidal agents. We have described the chemical synthesis of a range of CPPs conjugated to a 16-mer PNA that targets the HIV-1 TAR RNA apical stem-loop, which is situated at the 5'-end of all HIV-1 transcripts and which is the site of action of the trans-activator protein Tat.58 These were tested in an HeLa cell assay of inhibition of Tat-dependent trans-activation involving stably integrated luciferase-expressing plasmids. The activity depends on nuclear entry of the PNA. Stably linked conjugates (via a simple polyether linker) were unable to elicit significant inhibition of Tat-dependent trans-activation upon incubation up to 2.5 mM tested. However, two types of disulfide-linked PNA conjugate (Transportan or R6-Penetratin), when incubated for 24 hours, showed significant inhibitory activity (IC50 values of 0.5-1.0 mM), whereas conjugates of PNA with Tat (48-60) or Penetratin peptides failed to show activity unless chloroquine was co-administered.58

In parallel studies from the Pandey group, a similar disulfide-linked conjugate of a 16-mer PNA with Transportan elicited anti-HIV activity in chronically infected H9 cells by free delivery at 1-5 mM concentrations.93 Further, this Transportan-PNA conjugate showed high activity (IC50 ~ 50 nM) as a virucidal agent when pseudotyped vesicular stomatitis virus (VSV)-HIV virions were pre-treated with conjugate before CEM-cell infection.94 Similar levels of virucidal activity were found for several other CPP conjugates of the 16-mer PNA, but their corresponding antiviral activities were less good and IC50 values varied from 400 to 1100 nM.95 It is not known currently why virucidal activity is so high compared to antiviral activity, but the authors suggested that the PNA-peptide conjugates may enter HIV-1 virions and inhibit reverse transcription of HIV-1 viral RNA. CPP-PNA conjugates may, therefore, have most potential for development as virucidal agents. Perhaps HIV-1 RNA undergoing reverse transcription within an infected cell cytosol or RNA being transcribed Tat-dependently from the HIV-1 pro-virus in the cell nucleus may present a less readily accessible TAR target as occurs in virion RNA.

Robaczewska et al.,96 have shown that an unconjugated 15-mer PNA directed to the viral encapsidation signal of duck hepatitis B virus was able to decrease the viral genomic DNA by 30% in infected duck primary hepatocytes, compared to mismatch controls. Upon conjugation to an oligoarginine (R7) peptide the construct was able to decrease the viral DNA genome by 65% under the same experimental conditions. However, an NLS-PNA conjugate did not improve antiviral activity compared to PNA alone.

It was recognized from the very early days that pyrimidine-rich PNAs can strand-invade double-stranded DNA containing complementary purine-rich sequences to form double strands or triple strands with two PNA strands and one DNA strand.97 The subsequent development of this approach to the targeting of double-stranded DNA and chromosomal DNA within cells has been reviewed recently.98 For example, a mixed sequence 17-mer anti c-myc PNA conjugated to the NLS peptide PKKKRKV by stable linkage exerted steric block antigene activity through downregulation of c-myc oncogene expression by interfering with transcription, but only at a relatively high concentration (10 mM).99 Perhaps more effective as a strand-invasion method is the use of bis-PNA linked via polyether linkage and attached to a cationic peptide D-(AAKK)4 with rates of invasion 100 times faster than bis-PNA not attached to the peptide.61

Another important study area is the bactericidal activity of PNA conjugated stably to a membrane-active peptide KFFKFFKFFK.57,58 In some cases, PNA targeted to ribosomal RNA or mRNA was able to cure HeLa cell cultures of bacterial infections. Although toxicity of the peptide in in vivo studies has precluded industrial development of this PNA-peptide conjugate, modified and less toxic peptides are now being used in the commercial development of PMO conjugates.100

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