A stearylated CPP for delivery of splice correcting oligonucleotides using a non-covalent co-incubation strategy

Aberrations in splicing patterns play a significant role in several diseases, and splice correction, together with other forms of gene regulation, is consequently an emerging therapeutic target. In order to achieve successful oligonucleotide
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  A stearylated CPP for delivery of splice correcting oligonucleotides using anon-covalent co-incubation strategy Maarja Mäe ⁎ , Samir EL Andaloussi, Per Lundin, Nikita Oskolkov, Henrik J. Johansson,Peter Guterstam, Ülo Langel Department of Neurochemistry, the Arrhenius Laboratories for Natural Sciences, Stockholm University, Svante Arrheniuväg 21A, SE-10691 Stockholm, Sweden a b s t r a c ta r t i c l e i n f o  Article history: Received 17 September 2008Accepted 27 November 2008Available online 6 December 2008 Keywords: Cell-penetrating peptideCo-incubationPhosphorothioate 2 ′ -O-methyl RNASplice correctionStearylation Aberrations in splicing patterns playa signi fi cant role in several diseases, and splice correction, together withother forms of gene regulation, is consequentlyan emerging therapeutic target. In order to achieve successfuloligonucleotide transfection, ef  fi cient delivery vectors are generally necessary. In this study we present onesuch vector, the chemically modi fi ed cell-penetrating peptide (CPP) TP10, for ef  fi cient delivery of a splice-correcting 2 ′ -OMe RNA oligonucleotide. Utilizing a functional splice correction assay, we assessed thetransfection ef  fi ciency of non-covalent complexes of oligonucleotides and stearylated or cysteamidated CPPs.Stearylation of the CPPs Arg9 and penetratin, as well as cysteamidation of MPG and TP10, did not improvetransfection, whereas the presence of an N-terminal stearyl group on TP10 improved delivery ef  fi ciencyremarkablycomparedtotheunmodi fi edpeptide.Thesplicecorrectionlevelsobservedwithstearyl-TP10areinfact in parity with the effects seen with the commercially available transfection agent Lipofectamine ™ 2000.However,theinherenttoxicityassociatedwithcationiclipid-basedtransfectionscanbecompletelyeliminatedwhen using the stearylated TP10, making this vector highly promising for non-covalent delivery of negativelycharged oligonucleotides.© 2008 Elsevier B.V. All rights reserved. 1. Introduction The majority of human genes undergo alternative splicing, a keypost-transcriptional modi fi cation that contributes to protein diversitythrough highly regulated splicing events. The disruption of normalalternative splicingpatternscanleadtovariousgeneticdisorderssuchas cystic fi brosis, Duchenne'smusculardystrophy, and β -thalassaemia(for a review, see [1]). Aberrations in alternative splicing pattern have also been observed in many cancer-related genes (thoroughlyreviewed in [2,3]), for instance BCL-X, MDM2, FGFR1, and FAS [3]. As a result of the association between alternative splicing andnumerous pathological conditions, splice correction, together withother forms of gene regulation [4], is gaining increasing attention as atherapeutic target [3]. These therapies are primarily based on the delivery of synthetic oligonucleotides (ONs), with mechanisms of action ranging from conventional antisense for down-regulation tosplice switching of aberrantly spliced genes using splice-correctingONs. There are, however, a number of obstacles that needs to besurmounted before advancing these ON-based therapies into theclinic. The plasma membrane of cells, for instance, constitutes animpermeable barrier for these large hydrophilic, as well as neutralpharmaceutically relevant macromolecules, preventing them fromgaining access to the interior of cells. As an implication, numerousdelivery vectors, both viral and non-viral, have over the years beendeveloped to facilitate cellular uptake. One such group of non-viralvectors that is increasingly utilized for the delivery of various cargoesis the cell-penetrating peptides (CPPs). The  fi rst CPP was discoveredmore than a decade ago [5], and since then these short cationic peptides have been exploited not only  in vitro  but also  in vivo  toconvey a number of cargoes ranging from small ONs to large proteinsand plasmids [6].One pivotal consideration when utilizing CPPs for delivery is thecellular fate and, consequently, the bioactivity of the cargo. Whendelivering for instance splice-correcting ONs, it is absolutely criticalthat the cargo reaches the cellularcompartment where splicing of thepre-mRNA occurs, i.e. the nucleolus. However, as a result of theprimarily endocytic uptake of CPPs [7 – 9], the cargo is commonlyretainedinthe endosomalcompartment withoutreachingneitherthenucleolus nor the actual cell interior.  In vitro  this obstacle can beovercome with the use of different lysosomotropic agents, e.g.chloroquine, but this approach is not optimal for future  in vivo applications. In order to promote endosomal escape and increase thetransfection ef  fi ciency of CPPs a number of different strategies havebeen employed, for example the fusion of the Tat peptide with thein fl uenza virus HA2 domain [10] and the development of a histidine-containing endosomolytic alpha helical penetratin analogue [11].  Journal of Controlled Release 134 (2009) 221 – 227  Abbreviations:  2 ′ -OMe ON, phosphorothioate 2 ′ - O -methyl RNA; Arg9, nona-arginine;CPP, cell-penetrating peptide; HKR, HEPES-buffered Krebs Ringer; ON, oligonucleotide;Pen, penetratin; TFA, tri fl uoroacetc acid; TP10, transportan 10. ⁎  Corresponding author. Tel.: +46 8 16 4266; fax: +46 8 16 1371. E-mail address:  maarja@neurochem.su.se (M. Mäe).       G      E      N      E      D      E      L      I      V      E      R      Y 0168-3659/$  –  see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.jconrel.2008.11.025 Contents lists available at ScienceDirect  Journal of Controlled Release  journal homepage: www.elsevier.com/locate/jconrel  Stearylation of CPPs has proven to be another successful methodol-ogy to increase the transfection ef  fi ciency of both plasmids [12] andsiRNA [13,14], through a non-covalent approach resulting in the formation of nanoparticle complexes. Another simple modi fi cationapproach is to introduce a C-terminal cysteamide, which appears to beimperativeforCPP-mediatedsiRNAdeliveryusingtheMPGpeptide[15].In this study, we evaluated the effect of N-terminal stearylation of three different cell-penetrating peptides – transportan 10 (TP10) [16],penetratin (Pen) [5], and nona-arginine (Arg9) [17] for the delivery of  splice-correcting phosphorothioate 2 ′ - O -methyl RNA (2 ′ -OMe ON)(Table 1) through a non-covalent co-incubation approach. In order toevaluate the transfection ef  fi ciency, we utilized the functional splicecorrection assay developed by Kole et al. in 1998 [18]. The assay is based on the HeLa pLuc 705 cell-line stably transfected with aluciferase-encoding gene interrupted by a mutated  β  -globin intron 2.This mutation causes aberrant splicing of luciferase pre-mRNAresulting in the synthesis of non-functional luciferase [18]. Masking the aberrant splice site with, for instance, an antisense 2 ′ -OMe ONredirects splicing towards the correct mRNA and consequentlyrestores luciferase activity. Our results show that stearylationincreases TP10-mediated splice correction approximately thirtytimes as compared to the unmodi fi ed peptide, reaching similartransfection levels as obtained with the commercially availabletransfection agent Lipofectamine ™ 2000. However, the stearylatedpeptide displays a substantially lower toxicity, making this peptide abetter alternative for ef  fi cient transfection. 2. Materials and methods  2.1. Synthesis of oligonucleotides and peptides Phosphorothioate 2 ′ - O -methyl RNA oligonucleotides were synthe-sized on an ÄKTA ™ oligopilot ™ plus 10 synthesizer with Oligosynt ™ 15(GE Healthcare, Sweden) pre-packed synthesis columns as previouslydescribed [19]. For 5 ′ -labeling, a molar excess of 10 equivalents Cy5amidite(AmershamBiosciences,USA)at0.1MwasusedandCy5amiditerecycling went on for 10 min. The molarity of ONs was determined byOD-measurements.The peptides Pen, Arg9, and TP10 were synthesized using  t  -Bocsolid-phase peptide synthesis strategy as previously described [20].Stearylated peptides were prepared by treatment of peptide resinswith3equivalentsstearicacidand3equivalents2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetra fl uoroborate (Nova ChemicalCompany, USA), 3 equivalents 1-hydroxybenzotriazole and 8 equiva-lents diisopropylethylamine (Fluka, Sweden) in dimethylformamidefor 30 min. C-terminally cysteamide modi fi ed TP10 was assembled byFmoc chemistry using cysteamine-2 chlorotrityl resin (CBL Patras,Greece) and the  fi nal cleavage was performed using tri fl uoroaceticacid (TFA) with 2.5% triisopropylsilane and 2.5% water. MPG peptidewas kindly provided by Frédéric Heitz (CRBM-CNRS, Montpellier,France).All peptides were puri fi ed by reversed-phase high-performanceliquid chromatography (RP-HPLC) on a Discovery ® C-18 Supelco ® column (Sigma-Aldrich, Sweden) using a gradient of acetonitrile/water containing 0.1% TFA. The identity of the puri fi ed products wasveri fi ed by analytical RP-HPLC and by Perkin Elmer prOTOF ™ 2000matrix-assisted laser desorption ionization time-of- fl ight mass-spectrometer (Perkin Elmer, Sweden). The mass-spectra wereacquired in positive ion re fl ector mode using  α -cyano-4-hydroxycin-namic acid as a matrix (Sigma-Aldrich) (10 mg/ml, 7:3 acetonitrile:water, 0.1% TFA). The molarity of the peptides was determined basedon dilutions of accurately weighed substances.  2.2. Cell culture HeLa pLuc 705 cells, kindly provided by R. Kole and B. Lebleu, weregrownat37°C,5%CO 2 inDulbecco'sModi fi edEagle'sMedium(DMEM)with glutamax supplemented with 0.1 mM non-essential amino acids,1.0 mM sodium pyruvate, 10% fetal bovine serum, 100 U/ml penicillinand 100 mg/ml streptomycin (Invitrogen, Sweden).  2.3. Complex formation 2 ′ -OMeONwasmixedwithCPPsatdifferentmolarratiosinHEPESKrebs Ringer (HKR) buffer (pH 7.4) in 10% of the  fi nal treatmentvolume (i.e. 30 µl). Complexes were formed for 1 h at roomtemperature and meanwhile the cell medium was replaced in 24-well plates to fresh serum free DMEM (270 µl). Thereafter complexeswere added to each well. When using Lipofectamine ™ 2000 (Invitro-gen, Sweden), the complexes were prepared according to manufac-turer's protocol. Complexes were analyzed by electrophoresis on a 6%agarose gel in TBE buffer, containing ethidium bromide (Sigma,Sweden), for 1 h at 100 V.  2.4. Quantitative cellular uptake Cells(100,000)wereseeded24hbeforetheexperimentin24-wellplates to reach a 70% con fl uence. Stearylated or unmodi fi ed peptidecomplexes with Cy5-labeled 2 ′ -OMe ON ( fi nal concentration 200 nM)at 5:1, 10:1 and 20:1 molar ratios were added to the cells in 300 µlserum-free DMEM. 1 h after treatment, cells were washed twice inHKR buffer and trypsinized for 10 minwith 0.25% trypsin (Invitrogen,Sweden). Cells were centrifuged at 1000 ×  g   for 5 min at 4 °C, and cellpellets were lysed in 250 µl 0.1 M NaOH for 1 h, after which 200 µllysate was transferred to a black 96-well plate. Fluorescence wasmeasured at 635/670 nm on FlexStation II  fl uorescence reader(Molecular devices, USA) and the amount of internalized compoundwas normalized to the amount of protein (Lowry, BioRad, USA).  2.5. Splice correction assay Cells (40,000) were seeded 24 h prior to experiments in 48-wellplates to reach 60% con fl uence 1 day post seeding. Cells were treatedwith peptide:2 ′ -OMe ON complexes at three different molar ratios(5:1, 10:1, and 20:1) for 4 h in serum-free media followed by theaddition of 300 µl 10% serum-containing medium and incubated foranother 20 h. Thereafter, the cells were washed and lysed using 50 µl0.2% Triton X-100 in HKR buffer for 30 min at room temperature.Luciferase activity was measured using Promega's luciferase assaysystem on GLOMAX ™ 96 microplate luminometer (Promega, Sweden)and normalized to protein content. Lipofectamine ™ 2000 (Invitrogen,Sweden) was used as a positive control for measuring transfectionef  fi ciency.In experiments with chloroquine, after complex formation andprior to treatment of cells, chloroquine ( fi nal concentration 100 µM)was added tothe transfection mixture in order topromote endosomalescape. 4 h after addition of the complexes and chloroquine to cells,  Table 1 Sequencesofcell-penetratingpeptides(CPPs)and2 ′ - O -methyl(OMe)oligoribonucleotideName Abbreviation SequencePenetratin Pen  a RQIKIWFQNRRMKWKK-NH 2 Transportan 10 TP10  a AGYLLGKINLKALAALAKKIL  b -NH 2 Nona-arginine Arg9  a RRRRRRRRR-NH 2 MPG MPG  c GALFLGFLGAAGSTMGAWSQPKKKRKV  b Phosphorothioate2 ′ -OMe RNA2 ′ -OMe ON  d 5 ′ -CCU CUU ACC UCA GUU ACA a N-terminal stearyl-modi fi cation. b C-terminal cysteamide modi fi cation. c N-terminal acetyl-modi fi cation. d Cy5 labeled.       G      E      N      E      D      E      L      I      V      E      R      Y 222  M. Mäe et al. / Journal of Controlled Release 134 (2009) 221 –  227   cell medium was replaced with fresh medium in order to avoidtoxicity effects.  2.6. Cell proliferation Wst-1 Cell proliferation was studied with the Roche Wst-1 proliferationassay according to the manufacturer's instructions. Brie fl y, 10,000cells/well were seeded 1 day prior to the experiment on a 96-wellplate in complete medium. Cells were treated with peptide:2 ′ -OMeON complexes at three different molar ratios (5:1, 10:1, and 20:1) for4 h in serum-free medium followed by the addition of 10% serumcontaining medium and incubated for another 20 h. Wst-1 was addedaccording to the manufacturer's protocol (Roche Diagnostics Scandi-navia AB, Sweden). Wst-1 measures the activity of the mitochondrialdehydrogenases to convert tetrazolium salts to formazan. Absorbancewasmeasuredat450nmonDigiscanabsorbancereader(LabvisionviaAH Diagnostics AB, Sweden). The percentage of viable cells wasdetermined using the GraphPad Prism software 4.0 (GraphPadSoftware, CA).  2.7. Statistics Values in all experiments are presented as mean±SEM of at least 3independent experiments done in duplicate. In the splice correctionassay with Lipofectamine ™ 2000 the differences between Lipofecta-mine ™ 2000 in complex with ON treated and untreated cells wereconsidered signi fi cant at **  p b 0.01 using ANOVA Dunnett's multiplecomparisontest.Inthesplicecorrectionassaythedifferencesbetweenchloroquine-treated and -untreated cells were considered signi fi cantat ***  p b 0.001 using ANOVA Bonferroni's multiple comparison test.Intergroup differences in the cytotoxicity test were consideredsigni fi cant at **  p b 0.01 using Student's  t  -test. 3. Results  3.1. Chloroquine signi  fi cantly increases unmodi  fi ed CPP-mediated splicecorrection Inordertocon fi rmthevalidityof thesplicecorrectionassayandtooptimize the oligonucleotide concentration, Lipofectamine ™ 2000was utilized to transfect the cells (Fig. 1A). The maximum splicecorrection was obtained with 200 nM ON, and virtually noluminescence was detected when excluding the transfection agent.The formation of non-covalent complexes between CPPs andvarious ONs has been reported previously by numerous groups, andthe complex formation has also been characterized using differentbiophysical techniques [12,21]. We qualitatively con fi rmed thepresence of the peptide:ON non-covalent complexes using an agarosegel retardation assay. TheON migrationwas severelyretardedbybothPen and TP10 at 10:1 and 20:1 molar ratios, whereas MPG displayed amore modest retardation (Supplementary Fig.1). Nevertheless, all theevaluated peptides form complexes with varying ef  fi ciency.Next, in line with the previously reported discrepancies betweenuptake and bioactivity [22], we demonstrate a weak correlationbetween the splice correction and the uptake of the differentunmodi fi ed CPP:ON complexes. In order to assess the uptake, a Cy5- Fig.1.  Unmodi fi ed CPP-mediated2 ′ -OMe ONuptake and splice correction.40,000 cells/well were seeded 24 h prior experiments in 48-well plates. Hela pLuc 705 cellswere treated with 2 ′ -OMe ON at four different concentrations in complex withLipofectamine ™ 2000 (A) or with unmodi fi ed peptide:2 ′ -OMe ON complexes at threedifferent molar ratios (5:1,10:1, and 20:1), (B) for 4 h in serum-free media followed byreplacementto10%serumcontainingmediumandincubatedadditionallyfor20h.Cellswerewashed with HKR buffer and lysed in 0.2% TritonX-100 and luciferaseactivitywasmeasured. (C) 100,000 cells/well were seeded 24 h prior experiments in 24-well plates.Cells were treated for 1 h with unmodi fi ed peptide:Cy5-2 ′ -OMe ON complexes at 5:1,10:1 and 20:1 molar ratios in 300 µl serum-free DMEM. Cells were washed with HKR buffer, trypsinized and centrifuged at 1000 ×  g  . Pellets were lysed in 0.1 M NaOH and fl uorescence was measured at 635/670 nm. Fluorescence was recalculated to pmolinternalized 2 ′ -OMe ON per mg protein. (D) Splice correction assay was carried out thesame wayas in A, the only difference is that 100 µM chloroquine (CQ) was added tothecomplexespriortocelltreatments.ONconcentrationin(B),(C)and(D)was200nM.Thevalues represent the mean of at least two independent experiments performed intriplicate (mean±SEM,  n =3). (A) **  p b 0.01, ANOVA Dunnett's multiple comparison test;(D) ***  p b 0.001, ANOVA Bonferroni's multiple comparison test.       G      E      N      E      D      E      L      I      V      E      R      Y 223 M. Mäe et al. / Journal of Controlled Release 134 (2009) 221 –  227   labelled 2 ′ -OMe ON was utilized in a quantitative  fl uorometric assay.DespitearathersubstantialuptakeofthePenandtheTP10complexes,the observed splice correctionwas only slightly higher than the splicecorrectionobtainedwhenusingonlytheON(Fig.1BandC).Moreover,the poorsplice-correcting results obtainedwith penetratin as avectorhas been documented previously [23], however, as a covalentconjugate. This probably indicates endosomal localization of thecomplexes, con fi rming that  fl uorescence-based methods, includingmicroscopy, rarely correlate with the delivery of bioactive cargo.Chloroquine had, as expected, a substantial impact on the splicecorrectionmediatedinparticularbyTP10 andPen(Fig.1D),despiteitsimpedingimplicationsforthequantitativeuptake(datanotshownand[24]). TheeffectsseenwhentreatingthecellssimultaneouslywiththeTP10:2 ′ -OMe ON complex and the lysosomotropic agent are in paritywiththesplicecorrectionobservedwhenusingLipofectamine ™ 2000.Pen induced splice correction to a lesser extent than TP10, whereasArg9didnotdisplayanysigni fi cantincreaseinspiteofthechloroquinetreatment (Fig.1D).  3.2. Cysteamide modi  fi cation did not have any signi  fi cant effects onsplice correction The presence of a C-terminal cysteamide has been reported to becrucial for cellular uptake and delivery ef  fi cacy of, among otherpeptides, the protein carrier Pep-1 [25] and the gene delivery systemMPG[15]. Consequently,wedecidedtoinvestigate thepossibleeffectsof theintroductionofacysteamidemodi fi cationonTP10andcomparethis with the previously described MPG peptide. Unsurprisingly, MPGinduced almost no splice correction at either ratio, which is inaccordance with previously published results [21]. On the other hand,TP10 with cysteamide modi fi cation was slightly more ef  fi cient,mediating a modest two-fold increase in comparison to untreatedcells at a 5:1 peptide:ON ratio (Fig. 2).  3.3. Stearylation of TP10 signi  fi cantly increases ON-mediated splicecorrection Asaresultofthesigni fi cantlyincreasedsplicecorrectionofthePenand the TP10 complexes when applying chloroquine treatment,suggesting that complexes are being retained in endosomal compart-ments, we decided to evaluate the previously described stearylationapproach [12 – 14] to improve the transfection ef  fi ciency of the splice-correcting ON. First, in order to determine the effects of stearylation,we assessed both the quantitative uptake and the splice correction of the N-terminally stearylated peptides (Fig. 3). Stearyl-TP10 andstearyl-Arg9 complexes displayed slightly higher uptakes than theunmodi fi ed peptides, whereas the inversed pattern was observed forPen (Fig. 3A). The quantitative uptake of the stearyl-TP10:Cy5-2 ′ -OMeON complex was approximately two times higher than the uptakemediated by TP10, but with a different ratio pattern (i.e. the 5:1 ratiowas taken up more ef  fi ciently than the 20:1 ratio). Stearyl-Arg9displayed approximately the same percentage increase as stearyl-TP10, but here the uptake pattern remained the same as for theunmodi fi ed peptides.Despitenodramaticdifferencesinthequantitativeuptake,stearyl-TP10 was considerably better at promoting splice correction than thetwo other stearylated peptides (Fig. 3B). Stearyl-TP10 at 5:1 and 10:1peptide:ONratiospromotedseveralordersofmagnitudehighersplicecorrection than stearyl-Pen and stearyl-Arg9 (Fig. 3B), in spite of thereports on stearylated octaarginine as a very ef  fi cient transfectionagent [12 – 14].The striking effects on splice correction with N-terminally steary-latedTP10areclearlyillustratedinFig.4.Stearylmodi fi cationimprovedsplice correction by approximately a factor thirty compared to theunmodi fi ed peptide, reaching values close to the splice correctionobserved when utilizing chloroquine in combination with unmodi fi edTP10. However, treating cells with stearyl-TP10 and chloroquinesimultaneously results in four times higher splice correction at a 5:1 Fig. 2.  Effect of cysteamide modi fi cation on splice correction. Hela pLuc 705 cells weretreated with cysteamide modi fi ed peptide:2 ′ -OMe ON complexes at three differentmolar ratios (5:1, 10:1, and 20:1) for 4 h in serum-free medium, followed byreplacement to 10% serum containing medium and incubated additionally 20 h. ONconcentrationwas 200 nM. Cells were washed with HKR buffer and lysed in 0.2% TritonX-100 and luciferase activity was measured. Splice correction assay results arepresented as fold increase over untreated cells. The values represent the mean of atleast three independent experiments performed in duplicate (mean±SEM,  n =3). Fig. 3.  Stearylated CPP uptake (A) and splice correction (B). (A) 100,000 cells/well wereseeded 24 h prior experiments in 24-well plates. Cells were treated for 1 h withstearylated peptide:Cy5-2 ′ -OMe ON complexes at 5:1, 10:1 and 20:1 molar ratios in300 µl serum-free DMEM. Cells were washed with HKR buffer, trypsinized andcentrifuged at 1000 ×  g  . Pellets were lysed in 0.1 M NaOH and  fl uorescence wasmeasured at 635/670 nm. Fluorescence was recalculated to pmol internalized 2 ′ -OMeON per mg protein. (B) Hela pLuc 705 cells were treated with stearyl-modi fi edpeptide:2 ′ -OMe ON complexes at three different molar ratios (5:1, 10:1, and 20:1), for4 h in serum-free medium followed by replacement to 10% serum containing mediumand incubated additionally 20 h. Cells were washed with HKR buffer and lysed in 0.2%Triton X-100 and luciferase activity was measured. ON concentration in bothexperiment was 200 nM. The values represent the mean of at least three independentexperiments performed in duplicate (mean±SEM,  n =3).       G      E      N      E      D      E      L      I      V      E      R      Y 224  M. Mäe et al. / Journal of Controlled Release 134 (2009) 221 –  227   ratio,indicatingthatalthoughthestearylmoietysigni fi cantlypromotesendosomalescape,asubstantialfractionremainstrappedinendosomes(Fig. 4). The decreased effects of chloroquine at higher molar ratios aremost likely a consequence of toxicity.In order to facilitate the comparison of the different transfectionsystems, unmodi fi ed and stearylated TP10, with or without chlor-oquine, was compared to Lipofectamine ™ 2000 (Fig. 5). The datashows that the ef  fi ciency of stearyl-TP10 is comparable withLipofectamine ™ 2000 while co-incubation of stearyl-TP10 withchloroquine is substantially better than the commercial transfectionreagent.  3.4. Stearyl-TP10 induces splice correction in a non-toxic fashion Non-toxic transfection reagents are naturally crucial in order topromote successful splice correction. To evaluate potential cytotoxiceffects of the peptides in complex with 2 ′ -OMe ON, the activity of mitochondrial dehydrogenases was measured as an indicator of healthy metabolically active cells. Neither the stearylated nor theunmodi fi ed TP10, Arg9, or Pen had any effect on cell proliferation ascompared to untreated cells (data not shown and Fig. 6). On the otherhand Lipofectamine ™ 2000 induced signi fi cant impairment of meta-bolic activity at the concentration suggested by the manufacturer,reducing the number of proliferating cells to 40% after 24 h. Inconclusion, stearyl-TP10 promotes the same level of transfection asLipofectamine ™ 2000 but without the detrimental cytotoxic effectsassociated with the cationic lipid. 4. Discussion The various different ON-based approaches for regulation of genefunction all have a common denominator in the inadequately lowbioavailability of the active biopolymers. Antisense and splice-correctingONs,siRNAs,andplasmidsallsharethisinherentweakness,with the implication that delivery vectors are absolutely pivotal foref  fi cient delivery. The increasing focus on ON-based therapies forvarious diseases has consequently resulted in an enhanced search foref  fi cient and non-toxic vectors for both  in vitro  and  in vivo applications. The use of CPPs to facilitate cellular uptake and toimprovethe general bioavailabilityof varioustypes of ONsholdsgreatpromise for the future. Initially, the default strategy for connectingCPPs to ONs (or other cargoes) was by covalent conjugation, usuallyaccomplished with the introduction of a reducible disul fi de bridge.However, a number of studies have recently emphasized the vitalityand versatilityof the co-incubation approach [26,27], where the cargoand the peptide simply is mixed at a certain molar ratio in order tocreate nanoparticle complexes capable of ef  fi cient cellular delivery.The signi fi cant role played by alternative splicing in severaldiseases has resulted in growing attention from both clinicians andresearchers. With the increasing knowledge regarding the mechan-isms behind deleterious splicing patterns, the possibility to correctthese genes has emerged as a feasible and attractive approach for themodulation of gene function. Various types of synthetic ONs, forinstance peptide nucleic acids [28,29], locked nucleic acids [19], and 2 ′ -OMe RNA [30], have been employed to correct aberrant splicingpatterns and their potency is generally initially evaluated using theabovementioned splice correction assay [18]. The versatility andpositive read-out of this functional model has made it the mainstayapproach for assessing both splice correction  per se  as well as theability of various vectors to deliver splice-correcting cargoes. In thepresentstudy,wedecidedtoutilizethisassaytoevaluatetheabilityof chemically modi fi ed CPPs to deliver splice-correcting 2 ′ -OMe ONusing the co-incubation approach.After having validated the speci fi city of the splice correction assayand optimized theONconcentrationusingthecommercialtransfectionagentLipofectamine ™ 2000,thevectorcapacityof theunmodi fi edCPPsTP10, Pen, and Arg9 was initially evaluated. Despite promoting aconsiderable quantitative uptake of a Cy5-labeled 2 ′ -OMe ON, neitherone of the peptides were able to mediate any signi fi cant splice Fig. 5.  Splice correction assay results are presented as fold increase over untreated cells atoptimalmolarratioforeachpeptide-ONcomplex(stearyl-TP1010:1,TP10+CQ10:1,stearyl-TP10+CQ 5:1) and compared to Lipofectamine ™ 2000. ON concentrationwas 200 nM. Fig. 6.  Cytotoxicity of peptide:ON complexes as compared to Lipofectamine ™ 2000.Toxicity was assessed by wst-1 assay 24 h after exposing cells to stearylated orunmodi fi ed TP10:2 ′ -OMe ON complexes at molar ratio 20:1, ON concentration was200 nM. The values represent the mean of at least three independent experimentsperformed in duplicate (mean±SEM,  n =3). Intergroup differences were consideredsigni fi cant at **  p b 0.01 according to Student's  t- test. Fig. 4.  Stearylated TP10 versus chloroquine treatment. Hela pLuc 705 cells were treatedwithunmodi fi ed,unmodi fi edwith chloroquine(CQ), stearylatedorstearylatedwithCQ TP10incomplexwith2 ′ -OMeONatthreedifferentmolarratios(5:1,10:1,and20:1),for4hinserum-freemedia followed byreplacement to10%serum containingmedium. ONconcentrationwas 200 nM. Chloroquine ( fi nal concentration 100 µM) was added to thecomplexes with unmodi fi ed TP10:2 ′ -OMe ON prior to cell treatment. Cells wereincubated additionally for 20 h and washed with HKR buffer and lysed in 0.2% Triton X-100 whereafter the luciferase activity was measured. The values represent the mean of at least three independent experiments performed in duplicate (mean±SEM,  n =3).       G      E      N      E      D      E      L      I      V      E      R      Y 225 M. Mäe et al. / Journal of Controlled Release 134 (2009) 221 –  227 
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