CLIP6-PNA-Peptide Conjugates: Non-Endosomal Delivery of Splice

Dec 6, 2017 - †The Institute for Drug Research, The School of Pharmacy and ‡Department of Biochemistry and Molecular Biology, Institute for Medica...
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Article Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

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CLIP6-PNA-Peptide Conjugates: Non-Endosomal Delivery of Splice Switching Oligonucleotides Terese Soudah,†,# Maxim Mogilevsky,‡,# Rotem Karni,‡ and Eylon Yavin*,† †

The Institute for Drug Research, The School of Pharmacy and ‡Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Faculty of Medicine, The Hebrew University of Jerusalem, Hadassah Ein-Kerem, Jerusalem 91120, Israel S Supporting Information *

ABSTRACT: Efficient delivery of oligonucleotides still remains a challenge in the field of oligonucleotide based therapy. Peptide nucleic acid (PNA), a DNA analogue that is typically synthesized by solid phase peptide chemistry, has been conjugated to a variety of cell penetrating peptides (CPP) as a means of improving its cellular uptake. These CPPs typically deliver their cargoes into cells by an endosomal-dependent mechanism resulting in lower bioavailability of the cargo. Herein, we designed and synthesized PNA−peptide conjugates as splice switching oligonucleotides (SSO) targeting the Mnk2 gene, a therapeutic target in cancer. In humans, the MKNK2 gene, is alternatively spliced, generating isoforms with opposite biological activities: Mnk2a and Mnk2b. It was found that the Mnk2a isoform is down-regulated in breast, lung, brain, and colon tumors and is a tumor suppressor, whereas MnK2b is oncogenic. We have designed and synthesized PNAs that were conjugated to either of the following peptides: a nuclear localization sequence (NLS) or a cytosol localizing internalization peptide (CLIP6). CLIP6-PNA demonstrates effective cellular uptake and exclusively employs a nonendosomal mechanism to cross the cellular membranes of glioblastoma cells (U87). Simple incubation of PNA− peptide conjugates in human glioblastoma cells up-regulates the Mnk2a isoform leading to cancer cell death.

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INTRODUCTION Splice switching oligonucleotides (SSOs) are short synthetic oligomers, typically 17−24-mers long that are designed to bind to a specific region of a pre-mRNA leading to modulation in splicing of a targeted gene. As the pre-mRNA should stay intact to allow the splicing event, the chemistry of SSOs is limited to steric blocking oligomers that include RNA modified oligomers such as 2′-OMe-RNA, 2′-MOE RNA, LNA (locked nucleic acid), and neutral synthetic oligomers such as phosphorodiamidate morpholino oligomer (PMO) and peptide nucleic acid (PNA).1 Using this approach, two SSOs have been recently approved by the FDA to treat two genetic disorders: duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA). In the first case, the drug (PMO) is given systemically. There has been much controversy regarding the approval of this drug and the FDA has requested to continue a larger clinical study in children suffering from DMD in order to provide more convincing clinical data on the efficacy of the drug (eteplirsen).2 For infants with the SMA disease, the drug (nusinersen) is injected directly to the CNS by ICV (intrathecal injection).3 These two drugs highlight the enormous potential of SSOs as therapeutic molecules but also show that systemic delivery of such molecules still remains a daunting challenge in the field of oligonucleotide based therapeutics. In recent years there has been much effort to improve the biological activity of these SSOs for the treatment of DMD. © XXXX American Chemical Society

Conjugation of peptides, termed cell penetrating peptides (CPPs), to PMO has shown great promise for the systemic delivery of PMOs to muscle cells (that do not produce dystrophin, the protein that is absent due to mutations/ deletions in the gene) including to cardiomyocytes in the heart.4−6 Peptide-PMOs were also recently shown to provide long-lasting in vivo activity in a mouse model for SMA after systemic (not ICV) administration.7 A variety of other genetic disorders (e.g., Usher syndrome, Myotonic Dystrophy, Cardiomyopathyrecently reviewed by Havens and Hastings8) have been studied as therapeutic targets using SSOs. The use of SSOs for treating cancer has been limited due to issues related to effective in vivo systemic delivery of SSOs. In vitro, a variety of oncogenes have been targeted by SSOs for exon skipping. For example, BRAC19 and Her210 genes were targeted by SSOs to induce exon skipping in breast cancer. In the former example, reduced expression of full length BRAC1 sensitized breast cancer cells to PARP inhibitors.9 In one study,11 the Bcl gene was targeted by 2′-MOE-PSRNA SSOs both in vitro and in vivo. This gene has two alternatively spliced transcripts: the anti-apoptotic Bcl-xL and the pro-apoptotic Bcl-xS. Using a nanodelivery system composed of liposomes and a cationic protein (protamine), Received: October 23, 2017 Revised: November 26, 2017

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DOI: 10.1021/acs.bioconjchem.7b00638 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry the authors were able to increase the Bcl-xS isoform leading to cancer cell death (in vitro) and to reduced cancer proliferation of a B16F10 tumor xenograft after systemic IV administration of the SSOs. In cancer, the switching of a splice variant by SSOs from an oncogene to a tumor suppressor may have an added therapeutic effect. Apart from the above-mentioned examples (e.g., Bcl), other genes may be promising candidates for such an approach. We have recently shown that the gene MKNK2, which encodes the enzyme Mnk2, is alternatively spliced, generating isoforms with opposite biological activities: Mnk2a acts as a tumor suppressor (in culture and in vivo) by binding to, phosphorylating, and activating the p38-MAPK stress pathway. Mnk2b on the other hand cannot bind to p38-MAPK but can still phosphorylate the translation initiation factor eIF4E which promotes cancer.12 In addition, we have shown that SSOs (2′OMe-RNA) that block the Mnk2b splice site and elevate Mnk2a, inhibit cancer cell growth and sensitize cancer cells to apoptosis.12 PNA delivery into cells and in vivo has been studies by several approaches. One approach relies on modification of the PNA itself. For example, guanidinium-PNA (GPNA)13,14 and γGPNA15 are PNA analogues that possess a positive charge (guanidine) on their backbone. This strategy has been successfully used to down-regulate EGFR expression in a cancer mouse model.16 Fluorinated PNAs were also shown to have improved cellular uptake in comparison to their nonfluorinated counterparts.17,18 Another approach relies on the use of nanoparticles for PNA delivery.19,20 Finally, the most straightforward and well-studied approach relies on the use of CPPs. A variety of CPPs such as Tat,21,22 Transportan,23,24 R6penetratin,1,25,26 peptide−dendrimer,27 peptide−lipid,28 PNA internalization peptides (pip),29 and M91830 have been conjugated to PNA and studied as antisense and SSOs. In many of these studies, an endosomal entrapment of the PNA was evidenced as the addition of an endosome-disrupting agent (e.g., chloroquine) resulted in increased biological activity of the PNA. A recent paper31 has shown that the (low) pH sensitive peptide (pHLIP) conjugated to a PNA targeting the miR-155 oncomiR was very effective in targeting this PNA in cancer cells as well as in a mouse model in vivo. This study highlights the great therapeutic potential of CPP-PNA conjugates. In this study, we have chosen a linear peptide termed CLIP632 (cytosol localizing internalization; KVRVRVRVDPPTRVRERVK, where DP is D-proline) as a potential CPP for the delivery of PNA that has not been explored thus far. CLIP6 has been shown to enter cells by a nonendosomal mechanism32 and to accumulate as the free peptide in the cytosol of cells. Accordingly, we have designed PNAs as SSOs targeting the MnK2a isoform. These PNAs were conjugated to two cell penetrating peptides (CPPs): (1) nuclear localization sequence (NLS) peptide, and (2) CLIP6. Both PNA−peptide conjugates were studied in cell culture (U87MG, glioblastoma) for their cellular uptake mechanism and the CLIP6 PNA conjugate was evaluated for its anticancer activity.

Scheme 1. Modulation of MnK2 Splicing by PNA−Peptide Conjugates

PNA anti2b (Table 1). Both Anti2b and scrambled PNA conjugates were synthesized on the solid support (see Experimental Procedures for details). The NLS peptide has been previously reported as an effective nuclear targeting agent.33−36 To the best of our knowledge, the NLS peptide has never been conjugated to a SSO as a means of targeting the SSO to the nucleus. In addition, we were very intrigued by the possibility of achieving a non-endosomal dependent uptake of the PNA SSO into cells by conjugating the PNA to CLIP6.32 This may result in higher biological activity due to the increased bioavailability of the PNA. U87MG cells are glioblastoma cancer cells that express high levels of the oncogenic splice variant Mnk2b (unpublished data). Therefore, we chose these cell lines to follow the splice switching activity of anti2b-PNA conjugates. U87MG cells were treated with anti2b PNA-conjugates by simple incubation of the SSO PNAs in cell culture. After 72 h, total RNA was isolated and cDNA was prepared from 1 μg of RNA using M-MLV reverse transcriptase. The two Mnk2 isoforms were then evaluated by RT-PCR (Figure 1). Mnk2a formation was clearly observed for Anti2b-PNA-CLIP6 (Figure 1). Interestingly, a decrease in the MnK2a isoform was evident at the higher dose of PNA SSOs (2.5 μM). It is possible that this reduction in splice switching activity is due to direct antisense activity of Anti2b-PNA-CLIP6 with the mature form of MnK2a in the cytoplasm. The splice switching was more pronounced for the Anti2b-PNA-CLIP6 conjugate in comparison to the NLS variant. Both scrambled PNA SSOs (with NLS and CLIP6 peptides) showed negligible effects highlighting the sequencespecific SSO activity. The splice switching activity of Anti2bPNA-CLIP6 was similarly observed in a repeated experiment (Figure 4S in Supporting Information). Given the higher splice switching activity of Anti2b-PNACLIP6, we focused on its effect on cell survival and proliferation. U87MG glioblastoma cells were seeded in 6well plates, and incubated for 24 h with 1.25 μM of Anti2bPNA-CLIP6 and its scrambled control. After 2 weeks, cell colonies were counted and photographed (Figure 2A). The graph represent colony numbers in the duplicate plates (Figure 2B). To examine the effect of PNA-CLIP6 SSOs on cellular transformation, we seeded U87MG glioblastoma cells into soft agar in the presence of Anti2b-PNA-CLIP6 and scrambled PNA-CLIP6 SSOs. Colonies were allowed to grow for 14 days. Colonies from 10 different fields were counted and the average

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RESULTS AND DISCUSSION PNA−peptide conjugates were designed to target the junction of intron 14 and exon e14b as shown in Scheme 1. This steric block is anticipated to increase the Mnk2a isoform (a tumor suppressor). Accordingly, we define our PNA constructs as B

DOI: 10.1021/acs.bioconjchem.7b00638 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry Table 1. Anti2b/Scr-PNA-NLS with/out FITC and Anti2b/Scr-PNA-CLIP6 with/out FITC Conjugates and Their Corresponding Maldi-TOF MS Assignmentsa name

description

construct N to C

mass (calculated)

mass (found)

Anti2b-PNA-NLS Scr-PNA-NLS Anti2b-PNA-NLS-FITC Scr-PNA-NLS-FITC Anti2b-PNA-CLIP6 Scr-PNA-CLIP6 Anti2b-PNA-CLIP6-FITC Scr-PNA-CLIP6-FITC

PNA-WT conjugated to NLS peptide PNA-Scr conjugated to NLS peptide PNA-WT conjugated to NLS peptide-FITC PNA-Scr conjugated to NLS peptide-FITC PNA-WT conjugated to CLIP6 peptide PNA-Scr conjugated to CLIP6 peptide PNA-WT conjugated to CLIP6 peptide-FITC PNA-Scr conjugated to CLIP6 peptide-FITC

GACTGTCCCACCTTCAGA-NLS ACGACCTTCTACGCCGCT-NLS FITC-Ahx-GACTGTCCCACCTTCAGA-NLS FITC-Ahx- ACGACCTTCTACGCCGCT-NLS GACTGTCCCACCTTCAGA-CLIP6 ACGACCTTCTACGCCGCT-CLIP6 FITC-Ahx-GACTGTCCCACCTTCAGA-CLIP6 FITC-Ahx- ACGACCTTCTACGCCGCTCLIP6

5752.79 5728.79 6253.70 6229.70 7029.28 7005.28 7530.40 7506.40

5749.00 5729.00 6262.00 6234.77 7030.44 7006.00 7535.86 7497.88

a

Ahx = 6-aminohexanoic acid.

Figure 1. Induction of Mnk2a isoform formation by Anti2b-PNA-NLS and Anti2b-PNA-CLIP6 SSOs. U87MG cells were incubated with 1.25 and 2.5 μM Anti2b-CLIP6-PNA, Anti2b-NLS-PNA or scrambled control PNAs. RT-PCR was determined after 72 h.

Figure 2. Anti2b-PNA-CLIP6 reduces colony survival of U87MG glioblastoma cells. (A) Colony formation assay of cells treated with 1.25 μM of Anti2b-PNA-CLIP6 and scrambled PNA-CLIP6 SSOs. After 2 weeks plates were fixed, stained, counted, and photographed. (B) Graph of the colony numbers in the duplicate plates.

C

DOI: 10.1021/acs.bioconjchem.7b00638 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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were incubated for 24 h with 1 and 5 μM Anti2b-PNANLS_FITC and Anti2b-PNA-CLIP6_FITC, respectively. Data provides evidence for effective cellular uptake of both PNA− peptide conjugates. However, the Anti2b-PNA-CLIP6_FITC has superior uptake as corroborated by FACS (Figure 5A). Given the effective and diffusive cellular uptake of the CLIP6 peptide,32 it was interesting to determine whether this property is still maintained after CLIP6 conjugation to the PNA SSO. Effective and direct PNA penetration of Scr-PNA-CLIP6_FITC to cells was provided by pretreating U87MG cells with inhibitors of endocytosis. First ATP depletion conditions37 were provided by treating cells with 5 mM sodium azide/25 mM 2-deoxy-D-glucose for 0.5 h prior to Scr-PNA-CLIP6_FITC (2 μM) addition and incubation (24 h). Here, we chose the scrambled PNA sequence as means of solely following cellular uptake without provoking a biological effect (splice switching activity). As shown in Figure 5B, a very small shift in FITC fluorescence was noted. Next, preventing clathrindependent mechanisms38 was achieved by incubating cells with 0.22 M sucrose prior to Scr-PNA-CLIP6_FITC (2 μM) addition and incubation (for 24 h). Figure 5B shows that inhibition of clathrin-dependent endocytosis did not change the overall amount of Scr-PNA-CLIP6_FITC internalized into cells. This suggests that energy-dependent mechanisms, like endocytosis, do not play a major role in Anti2b-PNACLIP6_FITC cellular uptake. It is likely that Anti2b-PNACLIP6_FITC employs nonendosomal mechanisms to cross cellular membranes. In summary, we have shown Mnk2 splice modulation by PNA−peptide conjugates. Anti2b-PNA-CLIP6 shows effective cellular uptake by a dominantly non-endosomal mechanism which results in higher bioavailability of the PNA SSO in the cytoplasm and nucleus (Figures 4 and 5). Indeed, a higher effect was achieved on splice switching of Mnk2 by Anti2bPNA-CLIP6 in comparison to the NLS version of this PNA (Figure 1). This splice switching activity is on par with colony survival and soft agar assays (Figures 2 and 3) confirming the tumor suppressor activity of the Mnk2a isoform.

number of colonies per well was calculated (Figure 3). A substantial decrease in colony formation was observed for

Figure 3. Anti2b-PNA-CLIP6 SSO reduces colony survival and inhibits colony formation in soft agar of U87MG glioblastoma cells. Colony formation assay of cells treated with Anti2b-PNA-CLIP6 SSO (1.25 μM). Cells were seeded in soft agar in duplicates.

Anti2b-PNA-CLIP6 providing further support for the anticancer activity exerted by Anti2b-PNA-CLIP6 as a consequence of elevating the levels of the tumor suppressor Mnk2a isoform. To explore PNA SSOs uptake and distribution into cells, both PNAs (Anti2b-PNA-NLS and Anti2b-PNA-CLIP6) were labeled with fluorescein isothiocyanate (FITC). Live cell imaging of Anti2b-PNA-NLS_FITC and Anti2b-PNA-CLIP6_FITC in U87MG cells are shown in Figure 4. Cells nuclei were stained with DAPI. Clearly, Anti2b-PNA-CLIP6_FITC shows a dominant fluorescence in the cytoplasm (Panel B, upper row) and the intensity of this signal is by far greater than that of Anti2b-PNA-NLS_FITC (Panel B, lower row). In fact, we find a ca. 3-fold increase in fluorescence intensity for Anti2b-PNACLIP6_FITC in comparison to Anti2b-PNA-NLS_FITC as quantified by Image-Pro Analyzer. To further probe PNA SSOs mechanism of uptake, a series of flow cytometry experiments were performed. U87MG cells

Figure 4. CLSM images of U87-MG glioblastoma cells incubated with FITC-labeled PNA SSOs. 1 μM FITC labeled Anti2b-PNA-CLIP6 (upper row) and Anti2b-PNA-NLS after 3 h incubation. (a) DAPI staining of nuclei, (b) FITC-PNA, and (c) overlay. D

DOI: 10.1021/acs.bioconjchem.7b00638 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 5. Cellular uptake of PNA SSOs as corroborated by FACS analysis. (A) 1 and 5 μM Scr-PNA-NLS_FITC and Scr-PNA-CLIP6_FITC were incubated for 24 h with U87MG cells. (B) 2.0 μM Scr-PNA-CLIP6_FITC were incubated for 24 h with U87MG cells (blue) and in the presence of NaN3/2-deoxy-D-glucose (green) and sucrose (light blue).

To the best of our knowledge, this is the first example of cellular delivery of PNA using a non-endosomal cell penetrating peptide (CLIP6).32 It remains to be determined whether this activity will translate into in vivo splice switching activity; a task that we are currently evaluating.

were triturated with cold diethyl ether, and the precipitate was collected. The PNA samples were analyzed on RP-HPLC (Shimadzu LC2010), using a semipreparative C18 reversephase column (Phenomenex, Jupiter 300 A) at a flow rate of 4 mL/min. Mobile phase: 0.1% TFA in H2O (A) and acetonitrile (B) (see Supporting Information, Figures S1−S2). The authenticity of each PNA−peptide conjugate was confirmed by MALDI-TOF mass spectrometry (Table 1). Final solutions were measured at 260 nm and calculated based on the extinction coefficients of the nucleobases. Cell Culture. U87MG cell line was grown in DMEM supplemented with 10% (v/v) FCS, penicillin, and streptomycin. RT−PCR. Tissue samples were minced in TRI reagent (Sigma) using Next Advance Bullet Blender 24. Total RNA was isolated and cDNA was synthesized from 1 μg RNA using MMLV reverse transcriptase and oligo dT in a final volume of 25 μL according to the manufacturer’s instructions (Promega). PCR was conducted on 1 μL of cDNA by KAPA 2G Fast HS ReadyMix PCR kit (KAPA Biosystems). PCR conditions were as described in manufacturer’s protocol of ReadyMix with the addition of 5% (v/v) DMSO for 35 cycles. PCR products were separated on 2% agarose gels. Primer sequences used are described in Supporting Information (Table S1). Clonogenic Assay. Twenty-four hours post transfection 1000, 500, and 250 cells were seeded in duplicate in 6-well plates with 2 mL of media (DMEM, 10% FCS). After 10−21 days cells were fixed with 2.5% glutaraldehyde solution for 10 min, stained with 1% methylene blue solution, photographed, and counted. Anchorage-Independent Growth. Twenty-four hours post transfection 15 000 cells per well were seeded in duplicate in 6-well plates. Each well was coated with 2 mL of bottom agar mixture (DMEM, 10% FCS, 1% agar). After the bottom layer had solidified, 2 mL of top agar mixture (DMEM, 10% FCS, 0.3% agar) containing the cells was added. After this layer had solidified, 2 mL of media (DMEM, 10% FCS) was added. Plates were incubated at 37 °C with 5% carbon dioxide. Following 10−21 days, colonies from 10 different fields were counted and the average number of colonies per well was calculated. Imaging and Cellular Uptake in U87MG Cell Line. Twenty-four hours prior to PNA addition, U87MG were plated on an 8-well chamber, removable microscopy glass slide sterilized 15Pcs/Box (NBT New Bio Technology Ltd.), until reaching 70−80% confluence. Before adding the PNAs, the

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EXPERIMENTAL PROCEDURES Materials. Fmoc-PNA monomers were purchased from PolyOrg, Inc. (USA) and used as received. Amino acids (FmocLys-(BOC), Fmoc-Val-OH, Fmoc-Arg(pbf)-OH, Fmoc-D-ProOH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(OtBu)OH, and N-9-fluoromethoxycarbonyl chloride were purchased from GL Biochem (Shanghai) Ltd. 6-Aminocaproic acid was purchased from Acros Organics. Fluorescein isothiocyanate (FITC) was purchased from Chem-Impex Int’l Inc. Solvents and reagents for peptide chemistry were purchased from Biolab (Israel). Solid-Phase Synthesis of PNA−Peptide Conjugates. PNA SSOs were synthesized on the solid phase (as full constructs of peptide, PNA, with/out FITC) in a continuous manner thus avoiding the need of repurification. The SPS was based on literature procedures with some modifications described as follows: The first monomer Fmoc-Lys-(BOC) or Fmoc-Val-OH was coupled twice to the free hydroxyl groups of NovaSynTGA resin (Merck, Germany) using 10 equiv of the amino acid, 5 equiv of diisopropylcarbodiimide (DIC), and 0.1 equiv of 4-dimethylaminopyrimidine (DMAP) in dry DMF. Fmoc deprotection was done by treating resin with 20% piperidine in DMF for 10′ (×2) followed by washing with DMF and dichloromethane. For a 10 μmol scale synthesis on TGA-NovaSyn resin (loading−0.25 mmol/g): 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium (HATU, 40 μmol), hydroxybenzotrilazole (HOBT, 40 μmol), diisopropylethylamine (DIPEA, 80 μmol), and Fmoc-amino acids/Fmoc-PNA monomers (40 μmol) were mixed in dry DMF (0.4 mL) for 5′ and the solution was then added to the amine functionalized resin and mixed for 60 min. For FITC labeling, Fmoc-Ahx was introduced on the N-terminus of the PNA, followed by Fmoc deprotection and addition of 20 μmol FITC, 40 μmol DIEA in 0.2 mL DMF for 48 h (×2). The addition of a linker, Fmoc-Ahx-OH, between the PNA and FITC, prevents its binding to the α-NH position, thereby avoiding its elimination during sequence cleavage. The PNA−peptide conjugates and PNA−peptide conjugates labeled with FITC were deprotected and released from the resin by treatment with 90:10 (v/v) TFA/m-cresol for 3 h. The PNAs E

DOI: 10.1021/acs.bioconjchem.7b00638 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry medium was replaced and the cells were incubated (37 °C, humidified atmosphere containing 5% CO2) with 1 μM and 5 μM of Anti2b-PNA-NLS and Anti2b-PNA-CLIP6 in complete medium, over a period of 3, 14, and 24 h, followed by two washings with PBS. Thereafter, the cells were fixed with 4% formaldehyde (Biolab, Israel) and washed three times with PBS. For negative control, untreated cells were used. Mounting agent (DAPI Fluoromount-G) was used for nuclear staining and antibleaching (Southern Biotech, Birmingham, AL, USA). Cell fluorescence analysis was performed using a Zeiss LSM 710 confocal laser scanning system (Carl Zeiss Micro Imaging GmbH, Jena, Germany). Quantification of fluorescence intensity for confocal images was carried out by the ImagePro Analyzer software (Media Cybernetics version 7.0). Cellular Uptake and PNA-CLIP6 Mechanism. To determine the Anti2b-PNA-peptide uptake, flow cytometry studies were performed by plating 2.25 × 105 U87MG cells/ well prepared in 6-well plate and allowed to adhere overnight under normal culture conditions. The medium (DMEM, 10% FCS) was replaced and the cells were incubated (37 °C, humidified atmosphere containing 5% CO2) with 1 μM and 5 μM of Anti2b-PNA-NLS and Anti2b-PNA-CLIP6 in complete medium, over 24 h. The medium with PNA was removed, and the cells were washed with fresh medium. Then the cells were released from the wells with 400 μL of 0.25% trypsin-EDTA solution for 5′ at room temperature. Cells were collected into 1.5 mL polypropylene vials, then sedimented for 5 min at 4.186 × g at room temperature. The supernatant was discarded. The samples were suspended with 800 μL cold PBS, filtered by Falcon Cell Strainers, 70 μm Nylon, and analyzed by Beckman Coulter CytoFLEX Flow Cytometer Platform (488 nm excitation laser) with gating based on normalized fluorescence of untreated cells to evaluate the percentage of cells which internalized the fluorescently labeled PNA−peptides. To determine the PNA-CLIP6 mechanism, cells were pretreated with endocytosis inhibitors. Optimal inhibitor concentrations was determined by an MTT assay (see Supporting Information, Figure S3). Cells were depleted of intracellular ATP by adding 5 mM sodium azide/25 mM 2deoxy-D-glucose in media (DMEM, 10% FCS) over 0.5 h. Treating cells with 0.22 M sucrose for 0.5 h in media (DMEM, 10% FCS) afforded clathrin-mediated endocytosis inhibition. After treatments, cells were washed with PBS, followed by incubation of Scr-PNA-CLIP6_FITC (2 μM) for 24 h. Control cells were incubated with the PNA-CLIP6_FITC (2 μM) without prior addition of endocytosis inhibitors. Cell treatment and uptake mechanism was followed by FACS analysis as mentioned above, using two repeats for each experimental condition.

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ORCID

Eylon Yavin: 0000-0002-3527-3215 Author Contributions #

Terese Soudaha and Maxim Mogilevsky contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Israel Science Foundation (grant No. 480/13). E.Y. acknowledges the David R. Bloom Center for Pharmacy for financial support.

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ABBREVIATIONS PNA, peptide nucleic acid; NLS, nuclear localization sequence; CLIP6, cytosol localizing internalization peptide; CPP, cell penetrating peptide; SSO, splice switching oligonucleotide; PMO, phosphorodiamidate morpholino oligomer; DMD, Duchenne Muscular Dystrophy; SMA, Spinal Muscular Atrophy; ICV, Intracerebroventricular; pHLIP, pH low insertion peptide; MKNK2, MAP kinase interacting serine/ threonine kinase 2; MAPK, Mitogen-activated protein kinase type 1 and 2; p38-MAPK, p38 map kinase; eIF4E, Eukaryotic translation initiation factor 4e; WT, Wild type; SCR, Scrambled

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00638. HPLC chromatograms, primer sequences, RT-PCR, and MTT assay (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax:+972-2-6757574. Tel: +972-2-6758692. F

DOI: 10.1021/acs.bioconjchem.7b00638 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.bioconjchem.7b00638 Bioconjugate Chem. XXXX, XXX, XXX−XXX