As an alternative to covalent conjugation or to electrostatic or hydrophobic complexation of CPPs to antisense oligomers hybridization specific assembly using carrier PNAs offers greater flexibility. In order to explore in more detail the possibilities for developing such “carrier CPP-PNAs” for cellular delivery of antisense oligomers, we synthesized a series of carrier PNAs modified with different delivery ligands such as octaarginine and/or decanoic acid (), complementary to different positions within the antisense PNA oligomer (cPNA1, cPNA2, and cPNA3) (Scheme 1
). The well characterized antisense cargo PNA is targeted to an intronic aberrant splice site of the luciferase pre-mRNA in HeLa pLuc705 cells and the antisense activity measured as luciferase activation accomplished as a consequence of splicing correction and interpreted as a measure of (productive) cellular (nuclear) uptake efficiency.33
Four carrier PNAs complementary to the C-terminus 9 nucleobases of the antisense PNA and differently modified (Naked-cPNA1(9), (D-Arg)8
-Deca-cPNA1(9), Deca-cPNA1(9)) were tested for enhancing the cellular antisense effect of unmodified PNA (naked asPNA) (). Naked antisense PNA itself did not show any antisense activity over background (even at the highest concentration used (6 μM)). However, the activity was increased up to 8-fold at 2 μM in combination with a decanoyl-octaarginine carrier CPP-PNA ((D-Arg)8
-Deca-cPNA1(9)), while the analogous decanoyl carrier PNA (deca-cPNA(9)) did not show any significant improvement. These carrier PNAs were also tested in combination with a second decanoyl carrier PNA (Deca-cPNA2(9)) targeting the other half (N-terminus 9 nucleobases) of the antisense PNA, inspired by the previously reported improved cellular uptake by lipidic modification of CPP-PNA.33
However, the additional carrier decanoyl-PNA did not improve the effect of the other carrier PNAs. On the contrary a significant decrease of activity for the active carrier CPP-PNA ((D-Arg)8
-Deca-cPNA1(9)) was observed. The reason for the decreased activity with a combination of two carrier PNAs is not entirely clear, but it might be due to steric blocking of the antisense PNA by the bound carrier PNAs or altered uptake (mechanism). To challenge the first hypothesis, we chose an active antisense decanoyl-octaarginine-PNA ((D-Arg)8
-Deca-asPNA) and transfected this in complex with one or two unmodified carrier PNAs ((Naked cPNA1(9)) and (Naked-cPNA2(9)) at 2 μM (). These two carrier PNAs both dramatically inhibited the antisense effect around 80% and in combination the antisense activity was reduced to background level. This clearly suggests that dissociation of the carrier PNA(s) from the antisense PNA strand is a rate limiting step for the antisense activity. Nonetheless, the results also indicate that carrier CPP-PNAs may improve the (spontaneous) cellular uptake of unmodified antisense PNA without direct chemical conjugation to the CPP. In order to further explore the carrier PNA aided transfection strategy, these carrier PNAs were also tested for effects on antisense octaarginine CPP-PNA conjugates ((D-Arg)8
-asPNA)(). The antisense activity of the (D-Arg)8
-asPNA (at 2 μM) was improved up to 6-fold and 2-fold by (D-Arg)8
-cPNA1(9) and (D-Arg)8
-Deca-cPNA1(9), respectively, while the decanoic acid carrier PNA did not show any improvement, but rather showed inhibitory effects. Especially, decanoic acid PNA2 (Deca-cPNA2(9)) showed the most significant inhibition among the carrier PNAs. These results indicate that carrier CPP-PNAs can improve the cellular uptake of antisense CPP-PNA. Based on these findings, we decided to optimize the binding strength (affinity) between the antisense PNA and the carrier CPP-PNA by changing a sequence length of carrier PNAs.
Figure 1. Carrier PNA effect on different antisense CPP-PNAs. Relative antisense activity in HeLa pLuc705 cells of antisense PNAs hybridized to a carrier PNA1 (Naked-cPNA1(9), (D-Arg)8-cPNA1(9), (D-Arg)8-Deca-cPNA1(9), Deca-cPNA1(9)) and/or a carrier PNA2 (Naked (more ...)
We synthesized a new series of carrier CPP-PNAs of different PNA length (9–6 nucleobases) with octaarginine-decanoic acid modification (Deca-cPNA1(9–6)-(D-Arg)8) and tested their effect on PNA antisense activity. In combination with unmodified antisense PNA (), the longest carrier CPP-PNA with 9 nucleobases showed the highest antisense activity improvement (at least 20-fold at 6 μM), and the activity at 2 μM equals antisense the effect of (D-Arg)8-asPNA at 2 μM. However, this activity improvement was decreased with shorter carrier CPP-PNAs. This could suggest that a certain length of carrier PNA (hence certain binding strength) is required to form a sufficiently stable complex during cellular delivery. Then, we tested the effect of these carrier CPP-PNAs on the antisense activity of octaarginine (D-Arg)8-asPNA (). In contrast to the results with the unmodified antisense PNA, the antisense CPP-PNA showed higher antisense activity in combination with the shorter carrier CPP-PNAs. The shortest carrier CPP-PNA of 6 nucleobases ((Deca-cPNA1(6)-D-Arg)8) showed the highest improvement of the activity yielding almost 10-fold activation at 2 μM whereas no activation was seen at 6 μM. The reason for the opposite length dependency for unmodified antisense PNA and CPP-PNA is not clear at this stage, but most likely it is reflecting a delicate balance of sufficient stability of the cPNA-asPNA complexes (which exhibit thermal stabilities (Tm) ranging from 45–64°C (hexa- to nonamers) for delivery and adequate lability for dissociation inside the cell.
Figure 2. Effect of carrier PNA length. Relative antisense activity in HeLa pLuc705 cells of antisense PNAs hybridized to carrier CPP-PNA of different PNA length (9–6 nucleobases) modified with decanoyl-octaarginine (Deca-cPNA1-(D-Arg)8 (PNA2963, 2961, (more ...)
In order to explore further the structure activity relations of CPP carrier PNAs, we next studied a series of similar cPNAs modified only with octaarginine. In this case no clear length dependence was found and as before activation was seen at 2 μM (). Likewise moving the peptide to the N-terminal instead of the C-terminal of the PNA did not affect the enhancement activity.
Although the results so far () would indicate negative effects on antisense activity of a lipophilic carrier PNA, despite the clear positive effect on antisense activity upon covalent conjugation to CPPs in the CatLip approach,33
we decided to more systematically test the effect on the antisense activity of (D-Arg)8
-asPNA of a series Deca-cPNA2 (9–6 nucleobases)) complementary to the N-terminus of the antisense PNA. However, these carrier PNAs all showed inhibitory effects on the antisense activity of the (especially at high PNA concentration (6 μM) (Fig. S1
). The longest nonamer carrier PNA (Deca-cPNA2(9)) showed the highest inhibition, and the inhibition remained even with the shortest hexamer carrier PNA (Deca-cPNA2(6)). The reason for this inhibitory effect when supplied in trans (hybridization) rather than in cis (direct conjugation) is not clear, but because of this (unexpected) inhibitory effect, these carrier PNAs were not studied further.
Although CPP conjugation can very significantly improve PNA antisense activity, the efficacy of CPP-PNA conjugates may be further enhanced (up to 100-fold depending on CPPs and formulation) by endosomolytic agents such as chloroquine (CQ).34
In order to study this approach in relation to carrier CPP-PNAs, two of the most active carrier CPP-PNA constructs, cPNA1(7)-(D-Arg)8
were chosen. The results ( and ) show that the antisense activity of the CPP-asPNAs (D-Arg)8
-asPNA and (D-Arg)8
-Deca-asPNA were improved (up to 15-fold) by CQ treatment and the activation is significantly more pronounced at lower PNA concentrations (e.g., 2 μM for (D-Arg)8
-asPNA and 0.5 μM for (D-Arg)8
-Deca-asPNA). Interestingly, employment of carrier CPP-PNAs Deca-cPNA1(6)-(D-Arg)8
enhanced the antisense activity to virtually the same level as did CQ treated CPP-PNA at 0.5 μM and 1 μM, respectively. The decrease of activity at higher PNA concentrations (6 μM for (D-Arg)8
-Deca-asPNA) is ascribed to cellular toxicity of PNA conjugates33
). In addition to the two promising carrier CPP-PNAs, we also tested two carrier PNA constructs conjugated to oligo-histidine residues, the tetrahistidine (His)4
-cPNA3(8) and hexahistidine (His)6
-cPNA3(8) carrier PNAs, which could potentially provide endosomal disruption by the “proton sponge effect” of histidine residues in the acidic endosomal compartment as previously reported.35
However, no improvement was observed (in fact activity decreased) in contrast to apparent successes reported in the literature.36,37
Finally, we studied the effect of CQ treatment on carrier CPP-PNA enhanced PNA antisense activity. The results presented in show that additional CQ treatment does not further enhance the activity obtained with the antisense PNA alone in combination with CQ. In fact some inhibition is observed, especially when using CatLip type PNAs and at least some of this effect could be due to cellular toxicity under these conditions (Fig. S2
Figure 3. Oligohistidine carriers. Relative antisense activitiy in HeLa pLuc705 cells of antisense CPP-PNAs ((D-Arg)8-asPNA (PNA2787) and (D-Arg)8-Deca-as PNA (PNA2802)) hybridized to carrier CPP-PNA. Two carrier PNA constructs with oligohistidine ((His)4-cPNA3(8) (more ...)
Figure 4. Effect of chloroquine. Relative antisense activity in HeLa pLuc705 cells of two antisense CPP-PNAs ((D-Arg)8-asPNA (PNA2787) and (D-Arg)8-Deca-asPNA (PNA2802)) hybridized to a carrier CPP-PNA. Antisense PNA was hybridized with one of the carrier CPP-PNAs (more ...)
The luciferase data () were fully corroborated by mRNA splice correction analyses using RT-PCR (), and again these results demonstrate that the effect of carrier CPP-PNA delivery is comparable to or even higher than that obtained using CQ with the antisense PNA alone (compare for instance “no cPNA/+CQ with + cPNA1(7)-(D-Arg)8
/no CQ and Deca-cPNA1(6)-(D-Arg)8
/no CQ in ). These results suggest that carrier CPP-PNA-mediated enhancement of antisense CPP-PNA delivery could be an attractive alternative to auxiliary CQ treatment since this method gives comparable or even higher activity than that achieved by CQ treatment, without significantly increased cell toxicity (Fig. S2
), despite the cellular toxicity of (especially the CatLip) antisense CPP-PNAs observed at higher PNA concentrations (2-4 μM).
Figure 5. Effect of cholate carrier PNA. Relative antisense activity in HeLa pLuc705 cells of octaarginine conjugated antisense PNA ((D-Arg)8-asPNA (PNA2787)) hybridized to carrier PNA. Two carrier PNAs with backbone modification by Lys-derived backbone units ((Lys) (more ...)
Lipophilic ligands such as cholesterol and cholic acid2,38
have been found to increase cellular uptake and bioavailability of oligonuceotides, and especially cholic acid moieties were used to improve a lipid bilayer penetration of rather large hydrophilic compounds including 16 mer oligonucleotide,39
presumably by shielding of the hydrophilic cargo by a cholic acid “molecular umbrella.” Thus we synthesized a 10-mer carrier PNA containing three cholic acid moieties in the backbone of the PNA ((Cholate)3
-cPNA3(10), ). The activity of the antisense CPP-PNA ((D-Arg)8
-asPNA) together with (Cholate)3
-cPNA3(10) carrier at 2 μM was slightly higher (ca. 2-fold) than the antisense CPP-PNA itself while only a negligible increase was seen with a control carrier PNA construct without cholic acid moieties ((Lys)3
-cPNA3(10)). Thus the cholic acid modified carrier PNA did indeed enhance the antisense PNA activity, but to a significantly lesser degree than the carrier CPP-PNAs (up to 6-fold). The mechanism of the enhancement (improved membrane passage?) is not clear at this point.
In order to study the effect of carrier CPP-PNA on the cellular localization of the antisense CPP-PNAs by fluorescence microscopy, we synthesized fluorescein-labeled octaarginine antisense PNA (Fl-(Arg)8
-asPNA (PNA2919)) and used this for transfection of the cells in combination with carrier CPP-PNAs (cPNA1(7)-(D-Arg)8
). Fluorescence microscopy indicated a slightly higher intensity of fluorescence with more even distribution in both cytoplasm and nucleus upon delivery with carrier PNAs or upon CQ co-treatment as compared with treatment with the antisense PNA alone (Fig. S3
). The changes of the cellular localization of the cargo Fl-CPP-PNA by carrier CPP-PNAs were not as evident as suggested from the luciferase experiments. However, this preliminary experiment together with the luciferase experiments do suggest that carrier CPP-PNAs (like chloroquine) facilitate endosome disruption.
To further explore the carrier CPP-PNA delivery method for other (antisense) oligomers, we chose a single stranded short RNA originating from the antisense strand of an siRNA targeting normal luciferase mRNA. This single stranded antisense siRNA showed 70% downregulation of luciferase activity at 80 nM in p53R cells (expressing normal luciferase) when delivered by cationic lipid transfection (LFA2000) under conditions where the complete double stranded siRNA showed up to 90% downregulation (Fig. S4
). Two carrier CPP-PNAs targeting the PNA to the 3′-end 8 nt of the antisense siRNA strand and modified with either octaarginine alone ((D-Arg)8
-cPNA4) or with decanoyl-octaarginine ((D-Arg)8
-Deca-cPNA4) were synthesized. The antisense siRNA strand was hybridized to the carrier CPP-PNA at equimolar ratio and used for transfection in the absence or presence of CQ (). In the absence of CQ, the antisense RNA - alone or combined with either of the carrier CPP-PNA - did not show any significant luciferase downregulation even at the highest concentration (60 nM), while upon cationic lipid transfection very siginificant downregulation (90%) was seen. However, in combination with CQ treatment the decanoyl-octaarginine carrier CPP-PNA ((D-Arg)8
-Deca-cPNA4) very significant downregulation (59%) at 60 nM was obtained. This result emphasizes the general possibilities of exploiting carrier CPP-PNA based delivery formulation (via base-pair recognition) at low non-cytotoxic concentrations as it does not require a (large) molar excess of the cationic carrier, but also strongly suggests that delivery in this case occurs via endosomal pathways.
Figure 6. Downregulation of luciferase in p53R cells by a single stranded antisense siRNA (asRNA) delivered by carrier CPP-PNAs. Two octaarginine conjugated carrier CPP-PNAs (R8-cPNA, (PNA3164) and Deca-R8-cPNA (PNA3165)) with a complementary sequence to the asRNA (more ...)