Silencing of BCR/ABL Chimeric Gene in Human Chronic Myelogenous Leukemia Cell Line K562 by siRNA-Nuclear Export Signal Peptide Conjugates

Abstract

Herein we described the synthesis of siRNA-NES (nuclear export signal) peptide conjugates by solid phase fragment coupling and the application of them to silencing of bcr/abl chimeric gene in human chronic myelogenous leukemia cell line K562. Two types of siRNA-NES conjugates were prepared, and both sense strands at 5′ ends were covalently linked to a NES peptide derived from TFIIIA and HIV-1 REV, respectively. Significant enhancement of silencing efficiency was observed for both of them. siRNA-TFIIIA NES conjugate suppressed the expression of BCR/ABL gene to 8.3% at 200 nM and 11.6% at 50 nM, and siRNA-HIV-1REV NES conjugate suppressed to 4.0% at 200 nM and 6.3% at 50 nM, whereas native siRNA suppressed to 36.3% at 200 nM and 30.2% at 50 nM. We could also show complex of siRNA-NES conjugate and designed amphiphilic peptide peptideβ7 could be taken up into cells with no cytotoxicity and showed excellent silencing efficiency. We believe that the complex siRNA-NES conjugate and peptideβ7 is a promising candidate for in vivo use and therapeutic applications.

Introduction

Nucleic acid medicine, which can directly bind to the targeted gene and suppress its expression in a sequence specific manner, has been attracting intensive attention as revolutionary therapeutics based on a quite novel concept [1 –3]. Nucleic acid medicine can be expected to introduce the drastic treatment of HIV-1, late stage cancers, and any other incurable diseases. So far studied, currently commercialized nucleic acid drugs or promising candidates for commercialization include antisense oligonucleotide (ASO), siRNA, miRNA mimic, aptamers, DNA/RNA decoys, Ribozymes, and DNAzymes. ASO can bind to the complementary sequence of primary RNA transcripts, including mRNA, lncRNA, and miRNA guide strand in RNA induced silencing complex (RISC), and interferes their processing, translocation, and their native functions through an RNase H dependent cleavage or independent steric block. Exon skipping is one of the examples of modulation mRNA splicing [4 –7].

siRNA is a double stranded RNA with 21 nt length and can efficiently suppress mRNA or lncRNA by RISC dependent cleavage of the target in a sequence specific manner. Synthetic miRNA can enhance or suppress the function of endogenous miRNA by regulating mRNA or lncRNA in a RISC dependent manner. Ribozyme and DNAzyme function as sequence specific endonucleases against target primary transcript, mRNA and lncRNA. DNA/RNA aptamers and DNA/RNA decoys are usually screened through in vitro selection and function as competitive ligands or inhibitors for receptors, enzymes, transcription factors, RNA, DNA, and so on.

In the past decade and a half, a number of novel noncoding RNAs have been discovered by comprehensive analysis of transcripts and their crucial functions have been revealed step by step. It has been found that upregulation or downregulation of a specific miRNA expression is seriously related to various diseases, including cancers and neural disorders. lncRNA is another novel noncoding RNA involved in transcriptional and posttranscriptional regulation of genes, including epigenetic regulation. Therefore, the structure and the intracellular location of the target molecules for nucleic acid drugs are getting diverse more and more. The target molecules include genomic DNA, lncRNA, nuclear miRNA, and pre-mRNA in the nucleus and mRNA and miRNA in the cytoplasm. Proteins which contribute genetic regulation, metabolism, and signal transduction can be targets for DNA/RNA aptamers and decoys all over the cell.

Although medical applications of synthetic nucleic acids are extremely promising, a number of hurdles still need to be cleared to exert the full potential of them. Nucleic acid drugs should (1) be stable enough chemically and biologically, inside and outside the cells, (2) be delivered to the targeted tissue, (3) be taken up into cells effectively, (4) be localized in the proper intracellular site, (5) bind to the target molecule with high affinity and specificity, (6) not be toxic, (7) not cause undesirable effect (off-target effect), (8) not induce immune response, and (9) not interact with intracellular and extracellular molecules in a nonspecific manner [8 –12].

To clear these hurdles, chemical conjugation of oligonucleotide with biological and synthetic functional molecules by a covalent linkage has been attracting intensive attention more and more and now is recognized as one of the most powerful strategies along with chemical modification and noncovalent complexation [13 –16]. Oligonucleotide conjugates have been verified to improve the pharmacokinetic properties, the stability in vivo and in cells, the specificity of targeting tissues, the permeability into cells, the controlled intracellular trafficking with an aid of covalently linked peptides, lipids, sugars, protein ligands, RNA aptamers, antibodies, polyethylene glycol, and so on. Especially, since peptides derived from a great variety of proteins have extremely attractive and diverse structures and functions, oligonucleotide-peptide conjugates have been studied most intensely from a synthetic, a biological, and a medical point of view.

In our previous study, we reported the synthesis of oligonucleotide conjugates with nuclear localization signal (NLS) peptides, nuclear export signal (NES) peptides, designed peptides, and galactosamine by our original solid phase fragment coupling (SPFC) method [17]. SPFC is a convenient and versatile method and enables us to prepare DNA/RNA conjugates with peptides bearing arbitrary amino acid components and sequences. We successfully showed that oligoDNA-NLS peptide conjugates were taken up into cells without any transfection agent and localized in the cellular nucleus of human T lymphocyte cell line Jurkat. In contrast, oligoDNA-NES peptide conjugates were also taken up into Jurkat cells and localized in the cytoplasm outside the nucleus (Fig. 1). The controlled subcellular localization of oligonucleotides largely affected its antisense inhibition effect against human telomerase template RNA, that is, phosphorothioate DNA-NLS conjugate containing a peptide derived from Simian Vacuolating Virus 40 large T antigen (SV40 Tag) suppressed 99.6% of telomerase activity in 24 h and 95.3% in 48 h at 5 μM, whereas oligoDNA-NES conjugate derived from HIV-1 REV showed absolutely no inhibitory effect [18, 19].

FIG. 1.

Controlled intracellular localization of oligoDNA-signal peptide conjugates.

In the present study, we prepared two types of siRNA-NES conjugates by our original SPFC and evaluated silencing efficiencies of siRNA-NES conjugates against bcr/abl chimeric gene in human chronic myelogenous leukemia cell line K562.

Materials and Methods

General remarks

Oligonucleotides were assembled on controlled pore glass (CPG) support by automated DNA/RNA synthesizer nS-8 II (Gene Design, Inc.) using a standard cyanoethylphosphoramidite chemistry. Modification of 5′ end of oligonucleotides was performed with N-MMT-2-(2-aminoethoxy) ethyl phosphoramidite (Glen Research 5′-Amino Modifier 5). Fmoc protected amino acids for peptide synthesis were purchased from Nova Biochem and Watanabe Chemical Industries, Ltd. Peptides were synthesized by Biotage^® Initiator+ Alstra microwave assisted peptide synthesizer using a standard Fmoc chemistry on Wang-Resin (Nova Biochem). Oligonucleotides and conjugates were purified and analyzed by reversed phase high performance chromatography (RP-HPLC) on Hewlett-Packard HP-1100 system. RNA-NES conjugates and peptides were characterized by MALDI-TOF MS on Voyager-DE (Applied Biosystem) and ESI-MS on amaZon SL (Bruker Co.). Other chemicals for the synthesis of RNA-NEA conjugates were purchased from Wako, Kishida, Dojindo, and Aldrich. Total RNA extraction was performed with RNeasy Mini Kit (QIAGEN). Quantitative reverse transcription polymerase chain reaction was performed using SuperScript III Platinum One-step Quantitative RT-PCR System™ purchased from Life Technologies Co. on MX3005P (Agilent Technologies).

Peptide synthesis

Preparation of peptides was carried out by solid phase fmoc chemistry. The following side chain protected amino acids were used to obtain sufficient yield: Fmoc-Arg(Mtr)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, and other neutral amino acids were used as Fmoc-AA-OH. HOBt (N-hydroxybenzotriazole), TBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetra-methyluronium tetrafluoroborate), and DIEA (diisopropylethylamine) were used as coupling reagents for peptide elongation in dry N, N-dimethylformamide (DMF).

Peptide synthesis was carried out on Biotage Initiator+ Alstra microwave assisted peptide synthesizer, using Wang resin with a 0.05 mmol scale. The protocols of peptide synthesis included the following cycle: deprotection of Fmoc was carried out by 20% piperidine for 5 min (this procedure was repeated twice); the external piperidine was removed by DMF; five equivalents of amino acid, activated with TBTU and HOBt in 1 M DIEA, were coupled with amino acid assembled on the resin for 30 min; nonreacted amino acid was capped by anhydrous acetic acid. Cleavage and side chain deprotection (except for Lys(Tfa)) were accomplished with a mixture containing 1 M TMSBr/thioanisole in TFA with m-cresol/EDT for 2–8 h (depending on the peptide sequence), at room temperature with constant stirring. The crude peptide fragment was obtained by precipitation in cold ether. The crude peptide fragments were purified by RP-HPLC on oligoR3 (4.6/100 mm) peek column (PerSeptive Biosystems), at linear gradient from 100% A to 100% B for 45 min at a flow rate of 1.0 mL/min. Eluent A contains 0.1% trifluoroacetic acid in water, and eluent B contains 0.1% trifluoroacetic acid in acetonitrile. The products were characterized by RP-HPLC and MALDI-TOF MS. The data of RP-HPLC profiles and MALDI-TOF MS spectra of NES peptides P1 and P2 are shown in Supplementary Figs. S1 and S2; Supplementary Data are available online at www.liebertpub.com/nat.

P1: MALDI-TOF Mass m/z (M + H) theoretical Mw 1095.39, found 1095.87.

P2: MALDI-TOF Mass m/z (M + H) theoretical Mw 1278.54, found 1278.45.

Synthesis of RNA-NES peptide conjugates C1 and C2

The RNA-NES conjugates were synthesized by SPFC (Fig. 2). Oligonucleotides assembled on CPG (0.2 μmol scale) support in column using a standard cyanoethylphosphoramidite chemistry were reacted with 0.1 M N-MMT-2-(2-aminoethoxy)ethyl phosphoramidite in dry CH₃CN containing 1H-Tetrazole. 1H-Tetrazole was used as an activator to attach an amino group to the 5′ ends of the oligonucleotides. The column was washed five times by dry CH₃CN. MMT was removed by treatment of the column with 3% trichloroacetic acid (TCA) in dichloromethane. Then the amine was reacted with 50 eq of carbonyl diimidazole (CDI) in CH₃CN containing 4 eq of DIEA for 2 h at room temperature in dry conditions. CDI was chosen as a most appropriate linker in this study. The column was washed carefully with dry CH₃CN to remove external linker. Twenty equivalent amounts of peptide fragments, dissolved in 300 μL of dry DMF containing 10 eq of DIEA, were added to the CPG support, activated at 5′ end by CDI.

FIG. 2.

Synthesis of RNA-NES conjugates by solid phase fragment coupling. NES, nuclear export signal.

The CPG column, containing peptide fragment, was shaken for 24 h at 30°C under dry conditions and then washed thrice with 1 mL of DMF and five times with 1 mL of CH₃CN. The CPG column was dried by N₂-gas, and RNA-NES conjugates were cleaved and deprotected by treatment with concentrated aqueous ammonia for 4 h at 55°C in one step. RP-HPLC purification of RNA-NES conjugates was performed on an ODS column (5 μm, 4.6 × 150 mm) at 60 min linear gradient from 10% to 100% B at a flow rate of 0.2 mL/min. The mobile phase A contains 20 mM TEAA in water and phase B contains 20 mM TEAA in CH₃CN/H₂O (70/30).

In SPFC chemistry, the functional side chain of amino acid in peptide fragment could be free during conjugation, except for Lys(Tfa). Lys(Tfa) could be deprotected during the treatment of DNA/RNA-peptide conjugates assembled on CPG column with concentrated aqueous ammonia used for DNA cleavage and deprotection.

The products were characterized by RP-HPLC and MALDI-TOF MS. The data of RP-HPLC profiles and MALDI-TOF MS spectra of RNA-NES conjugates are shown in Supplementary Figs. S3–S6.

C1: MALDI-TOF Mass (M + H) theoretical Mw 7902.44, found 7901.99.

C2: MALDI-TOF Mass (M + H) theoretical Mw 8084.66, found 8085.16.

Cell culture and transfection

K-562 derived from chronic myelogenous leukemia was cultured in RPMI-1640 medium, supplemented with 10% heat-inactivated fetal bovine serum, streptomycin (100 μg/mL), and penicillin (100 U/mL) in a humidified atmosphere at 37°C over 5% CO₂. The cells used for transfections were at ∼70% confluence.

For transfection of siRNA-NES conjugates: 24–48 h before the treatment, the cell suspensions were cultured in 24-well plates (Iwaki, 1 mL cell suspension per plate) at a density 5 × 10⁵ cells/mL in RPMI-1640 medium without antibiotics. siRNA-NES conjugates were added to the plates in final concentrations of 1 and 5 μM, respectively. The mixtures were incubated for 24 and 48 h at 37°C in humidified atmosphere.

For transfection of siRNA-NES conjugates by Lipofectamine 2000™ (Invitrogen): siRNA-NES conjugates were diluted in 50 μL Opti-MEM (Invitrogen) and were incubated for 5 min at room temperature. Two microliters Lipofectamine 2000 were diluted in 48 μL Opti-MEM and incubated for 5 min at room temperature. Then both siRNA-NES conjugate and Lipofectamine 2000 solutions were combined together and incubated for 30 min at room temperature.

A hundred microliters of the RNA-NES conjugate-Lipofectamine 2000 complexes was added to 24-well plates, containing 900 μL of K562 cells (5 × 10⁵ cells/mL) in RPMI-1640 medium without antibiotics. The incubation was carried out following the manufacturer's instruction. The final concentrations of RNA-NES conjugates were 1 or 5 μM.

Quantitative analysis of BCR/ABL gene expression by real time reverse transcription polymerase chain reaction

Total RNA was extracted using RNeasy Plus Mini Kit (QIAGEN). Single step RT-PCR was performed using SuperScript III One-Step RT-PCR with Platinum^® Taq Kit (Invitrogen Life Technologies Corp.). The conditions for RT-PCR were as follows: 15 min at 50°C, 2 min at 94°C, and then followed by 40 cycles of amplification for 15 s at 94°C, 20 s at 55°C, and 20 s at 72°C. GAPDH was used as an internal control to ensure accuracy. The assay used an instrument capable of measuring fluorescence in real time (MX3005P; Invitrogen).

BCR/ABL mRNA Primers

Forward 5′-TGCAGATGCTGACCAACTCG-3′

Reverse 5′-GTTCCAACGAGCGGCTTCAC-3′

TaqMan Probe 5′-FAM-CAGTAGCATCTGACTTTGAGCCTCAGGGTCT-BHQ-3′

The results in Figs. 3 –8 are the means of triplicate determinations. Each determination was calculated from triplicate measurements. Each error bar indicates a standard deviation of the triplicate determinations.

FIG. 3.

Silencing of BCR/ABL in K562 by 5′-modified siRNA. siRNA mediated reduction of BCR/ABL mRNA expression in K562 cells. siRNAs were transfected by Lipofectamine 2000™. Normalized BCR/ABL/GAPDH mRNA levels were measured 24 h after transfection. The results are the means and standard deviations of triplicate determinations.

FIG. 4.

Silencing of BCR/ABL mRNA by siRNA5 (TFIIIA NES) in K562 siRNA mediated reduction of BCR/ABL mRNA expression in K562 cells. Normalized BCR/ABL/GAPDH mRNA levels were measured in 24 h after transfection by Lipofectamine 2000. The results are the means and standard deviations of triplicate determinations.

FIG. 5.

Silencing of BCR/ABL mRNA by siRNA6 (HIV1 REV NES) in K562 siRNA mediated reduction of BCR/ABL mRNA expression in K562 cells. Normalized BCR/ABL/GAPDH mRNA levels were measured in 24 h after transfection by Lipofectamine 2000. The results are the means and standard deviations of triplicate determinations.

FIG. 6.

Silencing of BCR/ABL mRNA by siRNA6 (HIV1 REV NES) transfected by Lipofectamine 2000 in K562 siRNA mediated reduction of BCR/ABL mRNA expression in K562 cells. siRNAs were transfected by Lipofectamine 2000. Normalized BCR/ABL/GAPDH mRNA levels were measured 24 h after transfection. The results are the means and standard deviations of triplicate determinations.

FIG. 7.

Silencing of BCR/ABL mRNA by siRNA6 (HIV1 REV NES) transfected by peptideα6 in K562 siRNA mediated the reduction of BCR/ABL mRNA expression in K562 cells. sRNAs were transfected by peptideα6. Normalized BCR/ABL/GAPDH mRNA levels were measured 24 h after transfection. The results are the means and standard deviations of triplicate determinations.

FIG. 8.

Silencing of BCR/ABL mRNA by siRNA6 (HIV1 REV NES) transfected by peptideβ7 in K562 siRNA mediated reduction of BCR/ABL mRNA expression in K562 cells. siRNAs were transfected by peptideβ7. Normalized BCR/ABL/GAPDH mRNA levels were measured 24 h after transfection. The results are the means and standard deviations of triplicate determinations.

Results and Discussion

Silencing of BCR/ABL chimeric gene by siRNAs bearing 5′-Amino Modifier 5

As a preliminary study to find out the desirable site for conjugation, before the synthesis of siRNA-NES conjugates, silencing efficiencies against bcr/abl chimeric gene were evaluated using siRNA1-4 bearing 5′-Amino Modifier 5 (Glen Research) at 5′ end of sense and/or antisense strand.

Target sequence of BCR/ABL mRNA (355–390)

5′-ggauuuaagcagaguucaa/aagcccuucagcggcca-3′

siRNAs (anti BCR/ABL mRNA361-381)

control siRNA (random): sense 5′-UCUCGCUUGGGCGAGAGUAATT-3′

antisense 3′-TTAGAGCGAACCCGCUCUCAUU-5′

siRNA1: sense 5′-GCAGAGUUCAAAAGCCCUUTT-3′

antisense 3′-TTCGUCUCAAGUUUUCGGGAA-5′

siRNA2: sense 5′-XGCAGAGUUCAAAAGCCCUUTT-3′

antisense 3′-TTCGUCUCAAGUUUUCGGGAA-5′

siRNA3: sense 5′-GCAGAGUUCAAAAGCCCUUTT-3′

antisense 3′-TTCGUCUCAAGUUUUCGGGAAX-5′

siRNA4: sense 5′-XGCAGAGUUCAAAAGCCCUUTT-3′

antisense 3′-TTCGUCUCAAGUUUUCGGGAAX-5′

X = −PO₄-CH₂CH₂OCH₂CH₂NH₂

Silencing efficiencies of control siRNA and siRNA1-4 were evaluated against BCR/ABL chimeric gene in human chronic myelogenous leukemia cell line K562 using quantitative RT-PCR normalized by GAPDH expression intensity (Fig. 3). Control siRNA with random sequence showed no effect, and siRNA1 as a positive control suppressed 59.8% of BCR/ABL gene expression at 200 nM and 53.7% at 50 nM. siRNA2 with X in the sense strand showed comparable silencing efficiency to unmodified siRNA1, while siRNA3 and siRNA4 with X in the antisense strand showed reduced silencing efficiencies. These results indicated that X in the sense strand did not influence a series of processes toward RISC formation, contacting to DICER, formation of RISC loading complex, loading to RISC, unwinding to matured RISC, and RISC activity. In contrast, X in the antisense strand hindered RISC formation and/or RISC activity. It can be interpreted that X in the sense strand did not decrease nor increase the ratio of incorporation of the antisense strand as a guide strand into RISC and that X in the antisense strand as a guide strand destabilized RISC after unwinding process because X would be protonated in water and electrostatically repel to the cationic residues in the MID domain of human Argonaute2 (Fig. 9) [20, 21]. These results strongly suggested that siRNA could be conjugated to a peptide at 5′ end of the sense strand without any serious loss of silencing efficiency.

FIG. 9.

5′-Terminus of the guide strand modified with X in the cationic pocket of MID domain of human Argonaute2.

Synthesis of RNA-NES conjugates C1 and C2

According to the results mentioned above, two types of NES peptides were covalently attached to the 5′ end of the sense strand of siRNA by SPFC as shown in Fig. 2.

The sense strand of siRNAs targeting to BCR/ABL mRNA (361–381) with two thymidines at 3′ end over hang region was assembled on CPG by standard automated protocols of cyanoethylphosphoramidite chemistry and modified at 5′ end with 5′-Amino Modifier 5 (Glen Research). After removal of MMT protection by 3% TCA, solution of N, N-diisopropylethylamine (DIEA) and CDI in absolute acetonitrile was added to the CPG column using a syringe at room temperature and left the column placed on a shaker for 2 h. Solution of 10 equivalents of a peptide fragment which was separately prepared by solid phase synthesis and purified by HPLC and 10 equivalents of DIEA in absolute acetonitrile was added to the CPG column using a syringe at room temperature and left the column stand for 24 h. The TFIIIA NES peptide fragment (NH₂CH₂CH₂CH₂CO-LPVLENLTL-OH) derived from Xenopus laevis [22 –24] used in the present synthesis has γ-aminobutyric acid (GABA) moiety with free reactive amino group at its N-terminus, and TFIIIA NES peptide fragment was linked to the 5′ end of RNA through GABA amino group. The HIV-1 REV NES peptide fragment (Ac-LPPLERLTL-KG-OH) [25, 26] has acetyl cap on N-terminal leucine and extra unprotected lysine and glycine at its C-terminus. HIV-1 REV NES peptide fragment was linked to RNA through ɛ-amino group of lysine with an adequate nucleophilic reactivity. The side chain of arginine contained in the NES peptide fragment derived from HIV-1 REV protein was set free during deprotection procedure using TFA before the conjugation step. The pKa value of DIEA is 10.76, which is slightly higher than the pKa value 10.67 of amino group of lysine and much lower than the pKa value 12.50 of guanidinyl group of arginine. That is why, it can be supposed that arginine side chain is protonated and has a decreased nucleophilicity, whereas lysine side chain is not protonated to undergo nucleophilic attack to isocyanate group in aqueous solution. In fact, HPLC profile of the isolated product was very sharp indicating a pure sole product. Cleavage from CPG support was performed with 28% aqueous ammonium hydroxide at 55°C for 4 h. The crude product was purified by reversed phase HPLC, and pure products were obtained in 26.8% for C1 (TFIIIA NES) and 31.2% for C2 (HIV-1 REV NES) isolated yields, respectively. The products were characterized by RP-HPLC and MALDI-TOF MS (Supplementary Figs. S3–S6).

C1: MALDI-TOF Mass (M + H) theoretical Mw 7902.44, found 7901.99.

C2: MALDI-TOF Mass (M + H) theoretical Mw 8084.66, found 8085.16.

Silencing of BCR/ABL chimeric gene by siRNA-NES conjugates

Using thus obtained two types of RNA-NES peptide conjugates C1 and C2, silencing efficiencies of siRNA5 and siRNA6 having C1 and C2 as a sense strand, respectively, were evaluated targeting 21 nt (361–381) in the junction region of b3a2 transcript from BCR/ABL chimeric gene in human chronic myelogenous leukemia cell line K562.

siRNA5: sense; NES-5′-GCAGAGUUCAAAAGCCCUUTT-3′

antisense; 3′-TTCGUCUCAAGUUUUCGGGAA-5′

NES; -HNCH₂CH₂CH₂CO-LPVLENLTL-OH (TFIIIA NES)

siRNA6: sense; NES-5′-GCAGAGUUCAAAAGCCCUUTT-3′

antisense; 3′-TTCGUCUCAAGUUUUCGGGAA-5′

NES; Ac-LPPLERLTL-K(ɛNH-)G-OH (HIV-1 REV NES)

As in Fig. 4 for siRNA5 and Fig. 5 for siRNA6, siRNA5 having C1 (TFIIIA NES) suppressed 91.7% of BCR/ABL gene expression at 200 nM and 88.4% at 50 nM, and siRNA6 bearing C2 (HIV-1 REV NES) suppressed 96.0% at 200 nM and 93.9% at 50 nM. Silencing efficiencies at lower concentrations of siRNA6 were clearly dependent on its concentrations while those of siRNA1 reached around 60% at 10 nM and did not increase at higher concentrations as shown in Fig. 5. Taking the results for siRNA1-4 and siRNA5-6 into account together, it is obvious that the distinguished increase of silencing efficiencies could be attributed to the presence of NES peptides. Previously we verified that oligoDNA-HIV-1 REV NES conjugate was taken up into human T lymphocyte cell line Jurkat and localized in the cytoplasm not in the nucleus [18, 19]. It can be reasonably presumed that the remarkable enhancement of silencing efficiency by siRNA-NES conjugates was owing to the cytoplasmic localization of the conjugates and efficient degradation of target mRNA. It should be pointed out that both NES peptides, one was linked to siRNA sense strand through amino group of N-terminal GABA and the other through ɛ-amino group of lysine close to C-terminus, indicated similar effects on RNA interference. And it is also speculated that modification of 5′ end of the sense strand with an NES peptide increased the chance of selection of the unmodified antisense strand as a guide strand in RISC. Unfortunately, siRNA-NES conjugates were not taken up into cells alone while oligoDNA-NES conjugates could penetrate into cells without any assistance of transfection reagents. siRNA-NES conjugates required the aid of commercial transfection reagent to penetrate into cells, and Lipofectamine 2000™ was used for the transfection. The reason why siRNA-NES conjugates could not penetrate into cells while oligoDNA-NES conjugates would enter into cells without any transfection reagents might be due to the double stranded structure of siRNA which has a rigid rod-like shape with more condensed negative charge than single stranded oligoDNA.

Transfection of siRNA-NES conjugates by designed peptides

We understand that nontoxic transfection of siRNAs into cells is one of the most crucial technologies to be established for in vivo use and medical applications. Next we explored transfection of siRNA-NES conjugates by designed peptides which could bind to and internalize RNA into cells. In our previous studies, we found out amphiphilic designed peptides which could bind to double stranded DNA and RNA [26 –29]. We investigated various types of designed peptides for the transfection of siRNA. We could find out that two peptides efficiently internalized the siRNA-NES conjugates into cells. One was named peptideα6 with a sequence of (LRAL)₆ which was known to form an α-helical structure in the presence of double stranded DNA or RNA, and the other was named peptideβ7 with a sequence of (RL)₇ which was known to form an antiparallel β-sheet structure in the presence of double stranded DNA or RNA. Transfection of siRNA-NES conjugates with 10–20 equivalent amounts of peptideα6 and peptideβ7 was carried out to be found successful. Silencing of BCR/ABL in K562 by the noncovalent complex of peptideα6 or peptideβ7 with siRNA6 was evaluated and the results are summarized in Figs. 6 and 7, respectively. The complex peptideα6-siRNA6 suppressed BCR/ABL expression up to 74.0% at 100 nM, while the complex peptideα6-siRNA1 suppressed bcr/abl expression only 19.8% at the same concentration. The complex peptideβ7-siRNA6 suppressed BCR/ABL expression up to 95.2% at 100 nM as high as siRNA6-Lipofectamine 2000 complex, while the complex peptideβ7-siRNA1 suppressed BCR/ABL expression only 30.2% at the same concentration. We observed that peptideα6- and peptideβ7-siRNA complexes were almost nontoxic to Jurkat and K562 cell lines and that both complexes were so resistant against nuclease digestions in 10% fetal bovine serum that half life time of siRNA in the complex was extended over 48 h, while that of naked siRNA was shorter than 1 h. More details of the transfection of siRNA by these designed peptides will be published elsewhere shortly.

Conclusions

Herein we successfully showed that siRNA-NES conjugates suppressed bcr/abl chimeric gene in human chronic myelogenous leukemia cell line K562 very efficiently. It can be reasonably speculated that NES peptide linked to 5′ end of sense strand of siRNA played a crucial role for the remarkable enhancement of silencing efficiency. Moreover we could show combination of siRNA-NES conjugate and peptideβ7 enabled nontoxic cellular uptake and excellent silencing efficiency. We believe that the combination is promising candidate for in vivo use and therapeutic applications.

Footnotes

Acknowledgments

This work was financially supported by JSPS KAKEN grant number 22550159. D.A.S. is supported by RSF grant no. 14-44-00068. A.A.F. acknowledges support from RFBR grant no. 16-03-01055.

Author Disclosure Statement

No competing financial interests exist.

References

van Ommen

G-JB

and Aartsma-Rus

. (2013). Advances in therapeutic RNA-targeting. N Biotechnol, 30:299–301.

Gavrilov

and Saltzman

. (2012). Therapeutic siRNA: principles, challenges, and strategies. Yale J Biol Med, 85:187–200.

Burnett

and Rossi

. (2012). RNA-based therapeutics: current progress and future prospects. Chem Biol, 19:60–71.

Koo

and Wood

. (2013). Clinical trials using antisense oligonucleotides in duchenne muscular dystrophy. Hum Gene Ther, 24:479–88.

Echigoya

and Yokota

. (2014). Skipping multiple exons of dystrophin transcripts using cocktail antisense oligonucleotides. Nucleic Acid Ther, 24:57–68.

Arechavala-Gomeza

, Khoo

and Aartsma-Rus

. (2014). Splicing modulation therapy in the treatment of genetic diseases. Appl Clin Genet, 7:245–52.

Spitali

and Aartsma-Rus

. (2012). Splice modulating therapies for human disease. Cell, 148:1085–1088.

Wang

, Liu

, Zheng

and Chen

. (2014). Rigid nanoparticle-based delivery of anti-cancer siRNA: challenges and opportunities. Biotechnol Adv, 32:831–43.

Lee

, Yoon

and Cho.

(2013). Recent developments in nanoparticle-based siRNA delivery for cancer therapy. Biomed Res Int, 2013:782041.

10.

Pan

, Thompson

, Meng

, Wu

, Xu

. (2011). Tumor-targeted RNA-interference: functional non-viral nanovectors. Am J Cancer Res, 1: 25–42.

11.

Wang

, Li

, Han

, Liang

and Ji

. (2010). Nanoparticle-based delivery system for application of siRNA in vivo. Curr Drug Metab, 11:182–96.

12.

Yin

, Kanasty

, Eltoukhy

, Vegas

, Dorkin

, and DG

Anderson

. (2014). Non-viral vectors for gene-based therapy. Nat Rev Genet, 15:541–55.

13.

Winkler

. (2013). Oligonucleotide conjugates for therapeutic applications. Ther Deliv, 4:791–809.

14.

Juliano

, Ming

and Nakagawa

. (2012). The chemistry and biology of oligonucleotide conjugates. Acc Chem Res, 45:1067–1076.

15.

Jeong

, Mock

, Oh

Y-K

and Park

. (2009). siRNA conjugate delivery systems. Bioconjugate Chem, 20:5–14.

16.

Dijk

, Venkatesan

and Kim

. (2006). Peptide conjugates of oligonucleotides: synthesis and applications. Chem Rev, 106:3712–3761.

17.

Kubo

, Morikawa

, Ohba

and Fujii

. (2003). Synthesis of DNA-peptide conjugates by solid-phase fragment condensation. Org Lett, 5:2623–2626.

18.

Kubo

, Zhelev

, Rumiana

, Ohba

, Doi

and Fujii

. (2005). Controlled intracellular localization and enhanced antisense effect of oligonucleotides by chemical conjugation. Org Biomol Chem, 3:3257–3259.

19.

Murao

and Fujii

. (2009). Organic synthesis and antisense effects of oligonucleotide-peptide conjugates. Curr Organ Chem, 13:1366–1377.

20.

Schirle

and MacRae

. (2012). The crystal structure of human argonaute2. Science, 336:1037–1040.

21.

Elkayam

, Kuhn

, Tocilj

, Haase

, Greene

, Hannon

and Joshua-Tor

. (2012). The structure of human argonaute-2 in complex with miR-20a. Cell, 150:100–110.

22.

Fridell

, Fischer

, Luhrmann

, Meyer

, Meinkoth

, Malim

and Cullen

. (1996). Amphibian transcription factor IIIA proteins contain a sequence element functionally equivalent to the nuclear export signal of human immunodeficiency virus type 1. Rev Proc Natl Acad Sci U S A, 93:2936–2940.

23.

Hanas

, Hocker

, Cheng

, Lerner

, Brackett

, Lightfoot

, Hanas

, Madhusudhan

and Moreland

. (2002). cDNA cloning, DNA binding, and evolution of mammalian transcription factor IIIA. Gene, 282:43–52.

24.

Ginsberg

, King

and Roeder

. (1984). Xenopus 5S gene transcription factor, TFIIIA: Characterization of a cDNA clone and measurement of RNA levels throughout development. Cell, 39:479–489.

25.

Malim

, McCarn

, Tiley

and Cullen

. (1991). Mutational definition of the human immunodeficiency virus Type 1 Rev activation domain. J Virol, 65:4248–4254.

26.

Fujii

, Hasegawa

, Niidome

and Aoyagi

. (1999). Enhancement of stability of double and triple stranded DNA by cationic amphiphilic α-Helix peptide. Nucleosides Nucleotides, 6:1623–1624.

27.

Fujii

, Hasegawa

, Niidome

and Aoyagi

. (1999). Thermal stability of double and triple stranded DNA in the presence of cationic amphiphilic α-helix peptide. Pept Sci, 1998:297–300.

28.

Yokoyama

, Kubo

and Fujii

. (2001). Amphiphilic β-sheet peptides can bind to double and triple stranded DNA. Nucleosides Nucleotides Nuc Acids, 20:1317–1320.

29.

Kubo

, Yokoyama

, Ueki

, Abe

, Goto

, Niidome

, Aoyagi

, Iwakuma

, Ando

, Ono

and Fujii

. (2001). Design, synthesis and characterization of DNA binding peptides. Pept Sci, 2000:109–112.

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