Cellular Uptake of Antisense Morpholino Oligomers Conjugated to

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Cellular Uptake of Antisense Morpholino Oligomers Conjugated to Arginine-Rich Peptides Hong M. Moulton,* Michelle H. Nelson, Susie A. Hatlevig, Muralimohan T. Reddy, and Patrick L. Iversen AVI BioPharma, Inc., Corvallis, Oregon 97333. Received December 5, 2003; Revised Manuscript Received January 14, 2004

Although the sequence specificity, biostability, and low toxicity of PMO (phosphorodiamidate morpholino oligomers) make them good antisense agents to study gene function, their limited ability to cross cell membranes limits their use in cell culture. In this paper we show that conjugation to arginine-rich peptides significantly enhanced the cellular uptake of PMO. The factors that affect the conjugate’s cellular uptake and its antisense activity toward a targeted mRNA were investigated. Factors studied include the number of arginines in the peptide, the choice of cross-linker, the peptide conjugation position, the length of the PMO, and the cell culture conditions. Delivery of PMO to the cell nucleus and cytosol required conjugation rather than complexation of peptides to PMO. R9F2C was best suited to deliver a PMO to its target RNA resulting in the strongest antisense effect. By simply adding the R9F2C-PMO conjugate into the cell culture medium at low µM concentration, missplicing of pre-mRNA was corrected. This particular peptide-conjugated PMO was more effective than the PMO conjugated to the transmembrane transport peptides of HIV-1 Tat protein, Drosophila antennapedia protein, or to peptides with fewer arginines. Length of PMO did not affect a peptide’s delivery efficacy, but all other factors were important. R9F2C peptide provided a simple and efficient delivery of PMO to a RNA target. Conjugation of peptide to PMO enhances the opportunities to evaluate gene functions in cell cultures.

INTRODUCTION

Phosphorodiamidate morpholino oligomers (PMO) (1, 2) are antisense molecules that inhibit gene expression by preventing translation or interfering with pre-mRNA splicing. The antisense specificity and efficacy of PMO have been well demonstrated in cells and in embryos (312). PMO are similar to DNA with two major structural differences: the negatively charged phosphorodiester internucleoside linkage in DNA is replaced by the neutral phosphorodiamidate linkage; the five-membered ring of deoxyribose in DNA is replaced by the six-membered ring of morpholine (Figure 1). The uncharged and hydrophilic PMO are highly resistant to enzymes and biological fluids (13). To bind to an RNA target, PMO must access the cytoplasm and/or nucleus. The typical length for a PMO to achieve effective antisense activity is between 18 and 25 bases. However, at this size, passive diffusion through cell membranes is slow. When using PMO to study gene function in cultured cells, delivery techniques such as scrape-loading (14) and using cationic lipid complexed with heteroduplexes of PMO/DNA (15) are generally required. Peptide-based delivery systems offer an attractive alternative to current delivery methods, being much simpler and relevant to in vivo applications. Some naturally occurring proteins, such as Drosophila antennapedia (Ant) and HIV-1 Tat, easily transport macromolecules into cells. The motifs within the transport proteins responsible for membrane “penetration” * Corresponding author: Hong M. Moulton, Ph.D., AVI BioPharma, Inc., 4575 SW Research Way, Suite 200, Corvallis, OR 97333. Phone: (541) 753-3635. Fax: (541) 754-3545. E-mail: [email protected].

Figure 1. Structure of DNA, PMO, and R9F2C-PMO conjugate. The structure of the conjugate is shown with R9F2C peptide conjugated at the 5′-end of PMO by the GMBS linker. “B” represents a base.

have been identified and characterized as cationic in nature (16-19). The cationic transport peptides are especially useful for delivering uncharged antisense oligomers due to lack of the problematic electrostatic interactions which may occur with anionic antisense oligomers. The electrostatic interactions between cationic peptides and anionic oligomers mask the positive charges of the peptide that are important for its delivery ability, subsequently decreasing the delivery efficacy of the peptide. Tat or Ant peptides have been used to deliver uncharged peptide nucleic acids (PNA) (19-22). PTD4 is an analogue of pTat peptide and was designed to have

10.1021/bc034221g CCC: $27.50 © 2004 American Chemical Society Published on Web 02/28/2004

Peptide-Mediated Delivery of Morpholino Oligomers

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Table 1. Peptides and PMO: Nomenclatures and Sequences names

sequence

length

Peptidesa pTat PTD4 pAnt R9F2C R6F2C R5F2C 705c 705-FLd 705(12)-FL 705(24)-FL

NH2-CYGRKKRRQRRR-CONH2 NH2-YARAAARQARAC-CONH2 NH2-DRQIKIWFQNRRMKWKKC-CONH2 NH2-RRRRRRRRRFFC-CONH2 NH2-RRRRRRFFC-CONH2 NH2-RRRRRFFC-CONH2 Antisense Oligomersb CCT CTT ACC TCA GTT ACA-acetyl CCT CTT ACC TCA GTT ACA-fluorescein CTT ACC TCA GTT-fluorescein CCT CTT ACC TCA GTT ACA ATT TAT-fluorescein

12 12 18 12 9 8 18 18 12 24

a The sequences of peptides are written using the standard one letter amino acid symbols from N to C terminus. b PMO sequences are written using the standard genetic code from 5′ to 3′-end. c The antisense PMO was targeted to a mutant splice site at the nucleotide 705 of human β-globin intron 2. d 705 had carboxylfluorescein attached at the 3′-end.

more R helical structure than pTat to improve cellular uptake (23). It has been reported that Jurkat cells take up the fluorescein-tagged PTD-4 33 times more than fluorescein-tagged pTat (23). We have previously shown that Tat peptide enhances cellular uptake of PMO in cultured cells (24) as well. But this requires high µM concentration to achieve antisense activity. Therefore, a more effective delivery peptide than the Tat peptide is needed. We designed a peptide containing nine arginines (R), two phenylalanines (F), and one cysteine (C) with the sequence of NH2-RRRRRRRRRFFC-CONH2 (R9F2C). Arginine residues in the Tat peptide are found to be crucial for the cell delivery; substitution of nonarginine residues in the Tat peptide sequence with arginines increases the cellular uptake of a fluorescein tag (25). We added two phenylalanines to R9 to increase hydrophobicity, enhancing interaction with cell membranes and improving purification by reverse phase chromatography. Adding a hydrophobic moiety onto an arginine-rich peptide has also been shown to increase uptake of the cargo (26, 27). A final cysteine residue provided a sulfhydryl group for conjugation to PMO. The effectiveness of a peptide to deliver PMO to the targeted mRNA was tested using a positive readout assay rather than conventional “knockdown” assays. Traditionally, decreasing gene expression of cells treated with antisense oligomers has been interpreted as the result of the antisense activity; however, other potential causes of decreased expression must be considered. For example, general suppression of gene expression in the target cells may result if an oligomer causes toxicity, if its degradation products are toxic, or if a delivery method causes toxicity. The toxicity may be misinterpreted as the antisense effect. To remedy these problems, an approach based on the ability of steric-blocking oligomers to alter mRNA splice site selection has been developed to evaluate antisense activity (28-30). Sensitive assays that give positive readout of antisense action, in the form of production of functional reporter protein, have been devised in both cell culture (31, 32) and in mice (33). These assays exploit mutations in intron 2 of human β-globin pre-mRNAs that lead to missplicing. The mutated intron cannot be properly excised resulting in an in-frame stop codon in the luciferase coding sequence. Two conditions must be met to produce the functional reporter protein at 37 °C. First, an antisense oligomer must enter the cell nucleus to block the mutated splice site, thus restoring normal splicing and read through of the reporter message. The result is mRNAencoding functional reporter protein. Second, but equally

important, the cells must be viable to carry out RNA processing and translation so that reporter protein activity is produced. Using the splice correction assay, we assessed the necessity of covalent attachment verses complexation of the peptide and PMO and compared the effectiveness of R9F2C to other peptides for delivery of PMO to the targeted pre-mRNA. We further determined the effects of conjugation chemistry, conjugation position, length of PMO, and cell culture conditions on the cellular uptake and antisense activity of peptide-PMO conjugates. The linker effects on intracellular distribution and concentration of the peptide conjugated PMO were also examined by fluorescent microscopy and flow cytometry. The cellular toxicity of the conjugates was evaluated. EXPERIMENTAL PROCEDURES

Peptides, PMO, and Cross-Linkers. The nomenclature and the sequences of peptides and PMO are listed in Table 1. All peptides were synthesized by Global Peptide Services (Ft. Collins, CO) and purified to >90% purity. PMO were synthesized at AVI BioPharma as described elsewhere (1, 34). Cross-linkers: Sulfo-EMCS (N-[-maleimidocaproyloxyl]sulfosuccinimide ester) was obtained from Molecular Bioscience (Boulder, CO). GMBS (N-[γ-maleimidobutyryloxy]succinimide ester), sulfoKMUS (N-[κ-maleimidoundecanoyloxy]sulfosuccinimide ester), SBAP (succinimidyl 3-[bromoacetamido]propionate), SPDP (N-succinimidyl 3-[2-pyridyldithio]propionate), and sulfo-LCSPDP (sulfosuccinimidyl 6-[3′-(2pyridyldithio)propionamido]hexanoate were acquired from Pierce (Rockford, IL). 3′-End Acetylated or Fluoresceinated PMO. Acetic anhydride (0.1 M), dissolved in N-methyl-2-pyrrolidinone (NMP) containing 1% N-ethylmorpholine (v:v) was added to PMO while the oligomer was still attached to the synthesis resin via the 5′-end. The PMO was incubated at 25 °C for 90 min, washed with NMP, cleaved off from the synthesis resin, and purified as described previously (24). The product was analyzed by MALDI time-of-flight mass spectrometry (MALDI TOF MS) and high-pressure liquid chromatography (HPLC). Seventy percent of the product corresponded to full-length sequence with acetyl at its 3′-end and a piperazine group at the 5′-end. 5-(and 6-) Carboxyfluorescein was attached to the 3′-end of PMO as described previously (24). Addition of a Cross-Linker to 5′-End of PMO. A cross-linker (Figure 2) predissolved in 50 µL of DMSO was mixed with a PMO (2-5 mM) dissolved in sodium phosphate buffer (50 mM, pH 7.2) containing 20% CH3CN at 1:2 PMO:cross-linker molar ratio. The mixture was

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Figure 2. Names and structures of the bifunctional cross-linkers.

stirred at 25 °C in the dark for 30 min, and the product was precipitated in 30-fold excess of cold acetone. The PMO-linker was lyophilized and analyzed by MALDITOF MS and HPLC. The conversion from PMO to PMOlinker was >90%. Peptide and PMO Conjugation. PMO-linker (2-5 mM) was dissolved in sodium phosphate buffer (50 mM, pH 6.5, 5 mM EDTA) containing 20% CH3CN. The peptide was added to the PMO-cross-linker solution at 2:1 peptide:PMO molar ratio. The reaction was stirred at 25 °C in the dark for 2 h. The conjugate was purified first by CM-sepharose (Sigma, St. Louis, MO) ion exchange chromatography to remove unconjugated PMO and then by reverse phase chromatography (HLB, Waters, Milford, MA) and lyophilized. The concentration calculation used for all conjugates was based on the total absorbance at 260 nm of a sample. As analyzed by MALDI-TOF MS and capillary electrophoresis, the final product contained about 60% peptide conjugated to the full length PMO with the balance composed of conjugates of shorter PMO, a small amount of unconjugated full, and shorter length PMO, and about 2% of unconjugated peptide. Conjugating a Peptide onto 3′-End of PMO. Addition of a cross-linker and conjugation to a peptide to 3′-end of PMO is described previously (24). Cell Culture. HeLa pLuc705 (32) cell line was obtained from Gene Tools (Philomath, OR). NIH3T3 cells were obtained from ATCC (Manassas, VA). Both cell lines were cultured in RPMI 1640 supplemented with 2mM glutamine, 100 µg/mL streptomycin, 100 U/mL penicillin, and 10% fetal bovine serum (FBS) (Hyclone, Ogden, UT) unless specified in the figure legends. Cell lysis buffer contained 10 mL of cell lysis reagent (Promega, Madison, WI) and one proteinase inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany). Fluorescent Microscopy. Exponentially growing cells were plated in a 48-well plate. The next day the conditioned medium was removed, and a test substance, in fresh medium, was added to the well. After a designated time, the cells were washed with phosphatebuffered saline (PBS) three times and imaged directly in the culture medium with a Nikon Diaphot 300 inverted microscope. Images were captured with an Olympus digital camera connected to a computer using MagnaFire software (Optronics, Goleta, CA). Dihydroethidium, a nucleic acid stain, was obtained from Molecular Probes (Eugene, OR). Flow Cytometry. HeLa pLuc705 cells, plated at 80 000 cells per well in a 24-well plate 20 h before the treatment, were treated with medium containing a test substance for various times at a specified concentration. After each treatment, cells were washed with PBS three times. To measure the internalized conjugate, the membrane-bound conjugate was removed by trypsinization (21, 35). Trypsin (100 µL) (0.25%, Hyclone, Logan, UT) was added to each well and incubated for 6 min at 37 °C, followed by addition of 300 µL of culture media. The cells were spun down and washed with PBS one time

then resuspended in 200 µL of a buffer containing PBS, 1% FBS, and 0.2% NaN3. The flow data (10 000 events) was collected using a BD FACSCalibur cytometer, and data was analyzed using FCS Express 2 (De Novo Software, Thornhill, Ontario, Canada). Correction of Misspliced Pre-mRNA. HeLa pLuc705 cells, plated in a 48-well plate 20 h before the treatment, were treated with medium containing a test substance at a specified concentration. At designated times, the cells were washed with PBS twice and the cell lysate was collected. Luciferase produced was determined by mixing 30 µL of cell lysate and 50 µL of luciferase assay reagent (Promega, Madison,WI) followed by measuring the light production using the Flx 800 microplate fluorescence/luminescence reader (Bio-tek, Winooski, Vermont). The relative light units were normalized to µg of protein determined by the bicinchoninic acid method following the manufacturer’s procedure (Pierce, Rockford, IL). Cytotoxicity. Cell viability was determined by an MTT (methylthiazoletetrazolium, Sigma, MO) assay. HeLa pLuc705 cells were incubated with or without a test substance for 24 h in a 96-well plate, the conditioned medium in each well was replaced with 100 µL of fresh medium containing 500 µg/mL MTT, and the cells were incubated for 4 h. The medium was removed, and DMSO was added to each well and mixed thoroughly. The absorbance was measured at 540 nm. The percent cell viability was calculated by normalizing absorbance of treated cells to the untreated cells. Statistical Analysis. Data were plotted and analyzed using GraphPad Prism 3.0 software (GraphPad Software, San Diego, CA). RESULTS AND DISCUSSION

Peptide-PMO Conjugates. The chemical structures of DNA, PMO, and R9F2C-705 conjugate are shown in Figure 1. The nomenclature, the linkers used, conjugation position, and calculated and observed mass and purity are tabulated (Table 2). Peptides and PMO were conjugated by bifunctional cross-linkers. The structures of cross-linkers are illustrated in Figure 2. The conjugate with the GMBS linker is designated as peptide-PMO, otherwise the name of a specific linker is included in the nomenclature. For example, the conjugate of R9F2C and 705-FL by GMBS or SPDP linker are designated as R9F2C-705-FL or R9F2C-SPDP-705-FL, respectively. Conjugation to R9F2C Peptide Was Required To Deliver PMO to the Targeted mRNA. The effect of R9F2C peptide on delivering PMO was first determined by comparing fluorescent images of HeLa pLuc705 and NIH3T3 cells treated with a fluorescein tagged PMO (705-FL), a mixture of free R9F2C and 705-FL (R9F2C +705-FL), and the conjugate of R9F2C and 705-FL (R9F2C-705-FL). To avoid artifacts that may be caused by cell fixation techniques (21, 35), all fluorescent images were taken of live cells where no fixative agent or mounting medium was used. Very little fluorescence was



Table 2. Peptide-PMO Conjugates: Nomenclatures, Linkers, and the Calculated and Observed Mass and Purity name

linker

calcd average mass

obsd average protonated massa

% purity of full length conjugate

pTat-705 PTD4-705 pAnt-705 R9F2C-705 R9F2C-705-FL R9F2C-EMCS-705-FL R9F2C-KMUS-705-FL R9F2C-SPDP-705-FL R9F2C-LCSPDP-705-FL R9F2C-SBAP-705-FL R6F2C-705 R5F2C-705 R9F2C-705(12)-FL R9F2C-705(24)-FL 705-R9F2Cb

GMBS GMBS GMBS GMBS GMBS EMCS KMUS SPDP LCSPDP SBAP GMBS GMBS GMBS GMBS GMBS

7857 7616 8558 8015 8330 8360 8430 8255 8433 8276 8015 7391 6375 10330 8310

7858 7617 8559 8014 8330 8359 8431 8253 8442 8277 8015 7391 6377 10331 8311

60 65 60 59 60 61 67 60 64 50 61 60 64 67 62

a Observed mass accuracy ( 0.05% of calculated protonated mass. b All peptides were added to the PMO 5′-end except for 705-R F C 9 2 for which R9F2C was added to the 3′-end of PMO.

Figure 3. Conjugation to R9F2C peptide was required to deliver PMO to the targeted mRNA. (A) Fluorescent images (200× magnification) of live HeLa pLuc705 or NIH3T3 cells treated with test substance at 10 µM for 20 h. The test substances, as indicated above the images, were a mixture of free R9F2C peptide and fluorescein tagged 705 (R9F2C + 705-FL) (1:1 molar ratio) and R9F2C705-FL conjugate. (B) R9F2C-705 conjugate corrected misspliced pre-mRNA and upregulated the expression of luciferase reporter gene while R9F2C + 705 (1:1 molar ratio) or 705 alone did not. HeLa pLuc705 cells were treated with the test substance at indicated concentrations in medium containing 10% FBS for 24 h. Antisense activity is represented by luciferase activity, expressed as the relative light units (RLU) per µg protein. Each data point is an average ( the range of means of two independent experiments each consisting of a triplicate sample.

observed with 705-FL (image not shown) or R9F2C + 705FL (Figure 3A). Mixed diffused/punctate cytosolic and nuclear fluorescence was seen in 100% of the cells treated with the R9F2C-705-FL conjugate (Figure 3A), suggesting successful delivery of PMO to the cytosol and nucleus in both cell lines. The necessity of the covalent attachment of R9F2C to PMO was evaluated in the splice correction assay, which measures antisense activity of PMO in the nucleus and gives a positive luciferase reporter signal using the HeLa pLuc705 cell line. This cell line (32) is stably transfected with a plasmid carrying the luciferase coding sequence interrupted by a mutated human β-globin intron 2. Cells treated with PMO (705) alone or with a mixture of free R9F2C and 705 showed the same level of luciferase as cells treated with vehicle control (H2O). However, cells treated with the R9F2C-705 conjugate produced luciferase in a dose-dependent manner (Figure 3B), indi-

cating successful nuclear delivery of PMO. The results show that conjugation of R9F2C peptide to PMO was required for delivery to the nucleus. R9F2C Was More Effective in Delivering PMO Than pTat, PTD4, pAnt, and Peptides with Fewer Arginines. Using the splice correction assay and flow cytometry, we compared the effectiveness of R9F2C to pTat, PTD4, and pAnt to deliver PMO to the nucleus. PMO conjugated to R9F2C corrected splicing 4, 14, or 19 times more effectively than the PMO conjugated to pTat, pAnt, or PTD4, respectively (Figure 4A). Cells treated with R9F2C-705-FL had 3, 12, or 17 times more fluorescence than the ones treated with pTat-705-FL, pAnt705-FL, or PTD4-705-FL (Figure 4B), respectively. R9F2C, pTat, and PTD4 are peptides with the same length but differ in their sequences and number of arginines. pTat, pAnt, or PTD4 contains six, three, or three arginines, respectively, while R9F2C has nine


Moulton et al. Table 3. Effect of the Linker and Linking Chemistry on the Antisense Activity of the R9F2C and 705-FL Conjugatesa

Figure 4. R9F2C was more effective to deliver PMO than pTat, PTD4, pAnt, and peptides with fewer arginines. (A). Luciferase activity of HeLa pLuc705 cells treated with the 705 conjugated to R9F2C, pTat, R6F2C, R5F2C, pAnt, or PTD4 by the GMBS linker at 15 µM in the medium containing 10% FBS for 24 h. Each data point is an average ( the range of means of two independent experiments each consisting of a triplicate sample. (B). Flow cytometry measurement of mean fluorescence of HeLa pLuc705 cells treated with 2 µM of a conjugate for 3 h. The antisense activity or mean fluorescence of each conjugate relative to the vehicle control (H2O) is quantified above each bar.

arginines (Table 1). It has been suggested that argininerich peptides with different primary and secondary structures have a similar uptake mechanism (36). The number of arginines appears to be more important (25), and the structural variations have less effect on cellular uptake (37, 38). These reports are based on studies using fluorescein-tagged peptides; a quantitative comparison using a functional assay has not yet been reported until this study. To directly verify whether reducing the number of arginines decreases the peptide’s delivery efficacy, the cellular uptake of R9F2C-705-FL was compared to R6F2C705-FL and R5F2C-705-FL by both flow cytometry and the splice correction assay. The mean fluorescence decreased as the number of arginines decreased (Figure 4B). Relative to the vehicle control (H2O) treated cells, the cells treated with R9F2C-705, R6F2C-705, or R5F2C705 gave 56, 14, or 5.6-fold increase in luciferase production, respectively (Figure 4A). Lower mean fluorescence given by the conjugates with fewer arginines indicated less PMO delivered by the peptide, which correlated well with their lower antisense activity. We show here, for the first time by an antisense functional assay, that the number of arginines greatly influences the delivery effectiveness of the peptide. Antisense Activity Was Affected by Linker Length and Not by the Conjugation Chemistry. We examined the effects of linker length (bond length) and connectivity between PMO and R9F2C on PMO antisense activity. All linkers used in this study (Figure 2) were bifunctional cross-linkers, containing an amine-reactive N-hydroxysuccinimide (NHS) and a sulfhydryl-reactive moiety. To have a meaningful comparison of the linkers, the PMO or R9F2C peptide used was from one synthesis batch. The 5′-end of the PMO was conjugated at one end of a cross-linker through NHS chemistry. The opposite end of the cross-linker was conjugated to the sulfhydryl group of R9F2C by maleimide, bromoacetyl, or pyridyl disulfide chemistry. The effect of the linker length was determined by the splice correction assay, and the results are summarized in Table 3. The effect was explored by comparing R9F2C705-FL, R9F2C-EMCS-705-FL, and R9F2C-KMUS705-FL and by comparing R9F2C-SPDP-705-FL to

treatment

linker type

vehicle control (H2O) R9F2C-705-FL R9F2C-EMCS-705-FL R9F2C-KMUS-705-FL R9F2C-SMPB-705-FL R9F2C-SMCC-705-FL R9F2C-SBAP-705-FL R9F2C-SPDP-705-FL R9F2C-LCSPDP-705-FL

N/A thiomaleimide thiomaleimide thiomaleimide thiomaleimide thiomaleimide thioether disulfide disufide

linker antisense activity spacer RLU/µg protein (Å) (range) N/A 6.8 9.4 15.7 11.6 11.6 6.2 6.8 15.6

1 (0.1) 102 (4.9) 141 (4.3) 171(14.3) 123 (2.1) 86 (1.4) 98 (3.2) 109 (2.9) 181 (7.8)

a HeLa pLuc705 cells were treated with 10 µM of an indicated conjugate in the serum free medium for 6 h. The antisense activity was indicated by luciferase activity, expressed as RLU/µg protein. Each data point is an average ( range of means from two independent experiments each consisting of a triplicate sample.

R9F2C-LCSPDP-705-FL. The first three conjugates contained a thiomaleimide bond, but differed in spacing between the peptide and PMO. R9F2C-KMUS-705-FL (15.7 Å spacer) and R9F2C-EMCS-705-FL (9.7 Å) had 1.7 or 1.4-fold higher antisense activity, respectively, than R9F2C-705-FL (6.8 Å) (Table 3). The pyridyl disulfide chemistry was used to make R9F2C-SPDP-705-FL and R9F2C-LCSPDP-705-FL, resulting in each conjugate containing a disulfide bond. LCSPDP (15.6 Å spacer) is about 8.8 Å longer than the SPDP (6.8 Å) linker, and R9F2C-LCSPDP-705-FL was almost twice as effective in splice correction as R9F2C-SPDP-705-FL. Overall, the conjugates with longer linker length had greater antisense activity. The effect of conjugation chemistry was determined by comparing three conjugates, R9F2C-SBAP-705-FL, R9F2C-705-FL, and R9F2C-SPDP-705-FL, in the splice correction assay. The three conjugates each had the same linker spacing but differed in linking chemistry: thioether in R9F2C-SBAP-705-FL, thiomaleimide in R9F2C705-FL and disulfide in R9F2C-SPDP-705-FL. These conjugates had the same antisense activity despite their difference in conjugation chemistry. The above results show that the antisense activity of the PMO was increased with longer linkers but not affected by the conjugation chemistry. Increasing the backbone and side chain flexibility (37) of an argininerich peptide or an addition of a hydrophobic moiety (26, 27) onto a peptide have both shown to give better delivery of a fluorescein cargo. The higher antisense activity of the conjugates with longer chains of (CH2)n moieties may be the result of their greater flexibility and/or hydrophobicity than those with shorter linkers. It is also possible that a longer linker reduces steric interference for PMO binding to the targeted pre-mRNA. Effect of Linker Type on Intracellular Distribution and Concentration of the Peptide-PMO Conjugates. The intracellular distribution of the fluoresceintagged PMO (705-FL), conjugated to R9F2C via different linkers, was determined by fluorescent microscopy. All conjugates containing thiomaleimide or thioether bonds gave a similar fluorescent pattern: a bright fluorescent spot in addition to a dimmer diffused fluorescence throughout the cells, as represented by the image of cells treated with R9F2C-705-FL (Figure 5A). Green and red dual fluorescence staining (Figure 5B and 5C) reveals that the bright fluorescent spot was located outside of the nucleus as shown by cotreatment of cells with the conjugate and dihydroethidium, a cell-permeable nucleic stain that fluoresces red upon DNA intercalation. This


Figure 5. Effect of the linker type on intracellular distribution of the peptide-PMO conjugates. (A). Fluorescent image (200× magnification) of live HeLa pLuc705 cells treated with 10 µM of R9F2C-705FL or R9F2C-LCSPDP-705-FL for 20 h in RPMI with 10% FBS medium. (B and C). Dual fluorescence staining of HeLa pLuc705 cells treated with 2 µM of either R9F2C-705FL or R9F2C-LCSPDP-705-FL in opti-MEM medium for 24 h. 10 µM of dihydroethidium, a nucleic acid stain, was added to the cells during the last 90 min incubation period of the conjugate. The same field image of cells was taken (400× magnification) through the green channel (B) or the red channel (C) of the microscope.

bright fluorescent spot seen using R9F2C-GMBS-705FL was not observed with either disulfide-containing conjugates, R9F2C-LCSPDP-705-FL (Figure 5A and 5B) or R9F2C-SPDP-705-FL. Instead, the latter conjugates gave dimmer but more diffused fluorescence throughout the cytoplasm and nucleus than the thiomaleimide- or thioether-containing conjugates.


The effect of thiomaleimide or disulfide linker on cellular uptake was measured quantitatively by flow cytometry. Cells treated with R9F2C-KMUS-705-FL exhibited significantly higher fluorescence than R9F2CLCSPDP-705-FL (Figure 6). Uptake of R9F2C-LCSPDP705-FL was comparable to R9F2C-KMUS-705-FL at the 30 min timepoint but did not increase with longer treatment as did R9F2C-KMUS-705-FL. This could be explained by the intrinsic instability of the disulfide bond of R9F2C-LCSPDP-705-FL in the serum and/or the cleavage of the disulfide bond by glutathione at the cell surface (39). Cleavage of PMO from the peptide outside of the cell membrane would decrease the amount of PMO delivered. Despite the significant difference in intracellular concentration between R9F2C-KMUS-705-FL and R9F2CLCSPDP-705-FL, both conjugates gave nearly identical antisense activities (Table 3) in a dose dependent manner (data not shown). This suggests that the amount of internalized PMO available for the targeted pre-mRNA in the cell nucleus was similar. These interesting results can be analyzed in light of the difference in their linker types. R9F2C-LCSPDP-705-FL contained a disulfide bond that can be cleaved by cellular reducing agents such as glutathione in the golgi apparatus (39, 40), releasing PMO from the peptide. R9F2C-KMUS-705-FL contained a thiomaleimide bond that does not have known cleavage enzymes and so the PMO likely remained bonded to the peptide. If this is the case, the positive charges of R9F2C may make it easier for R9F2C-KMUS-705-FL to bind anionic cellular components other than the targeted premRNA. The result would be a decrease in effective PMO concentration, despite its higher intracellular concentration than that of R9F2C-LCSPDP-705-FL. The above explanation was supported by fluorescent images (Figure 5A and 5C). PMO delivered by the peptide with the disulfide linker diffused throughout the cells, similar to the distribution of scrape-loaded unconjugated PMO (14). PMO delivered by the peptide with the thiomaleimide linker gave more distinct and localized fluorescence outside nucleus (Figure 5A), similar to the fluorescent image of fluorescein-tagged R9F2C (data not shown). These results indicate that the delivery peptide had greater influence on subcellular distribution of PMO with a noncleavable linker compared to a cleavable linker.

Figure 6. Effect of the linker type on intracellular concentration of the peptide-PMO conjugates. Flow cytometry diagram of the Hela pLuc705 cells treated with 1 µM of R9F2C-KMUS-705FL or R9F2C-LCSPDP-705-FL for 0.5 (red line), 1 (blue line), 2 (purple line), 4 (green line), or 7 (yellow line) h. The black filled histogram represented the background fluorescence of vehicle control (H2O) treated cells.


Figure 7. PMO with R9F2C at its 5′-end had greater antisense activity than PMO with R9F2C at its 3′-end. Luciferase activity of HeLa pLuc705 cells treated with the 705 conjugated to R9F2C either at the 5′-end (R9F2C-705) or the 3′-end (705-R9F2C) at indicated concentrations in the medium containing 10% FBS for 24 h. Each data point is an average ( the range of means of two independent experiments each consisting of a triplicate sample.

PMO with R9F2C at the 5′-End Had Higher Antisense Activity Than the PMO with R9F2C at the 3′End. Either the 3′-end or the 5′-end of the PMO can be conjugated to the peptide. The effect of position of the peptide on PMO was determined by comparing two conjugates of 705 with R9F2C either at the 3′-end (705R9F2C) or at the 5′-end (R9F2C-705). R9F2C-705 produced nearly four times as much as luciferase as 705R9F2C (Figure 7). The data indicate that the 5′-end of PMO is preferred for conjugation and can be understood in terms of PMORNA binding. PMO interrupt mRNA processes by steric blocking the access of other biomolecules to the target region (2). Addition of bulky moieties such as carboxylfluorescein or cholesterol to the 3′-end of a PMO decreases its antisense activity (data not shown), probably due to steric interference of the PMO/mRNA binding. We hypothesize that the positively charged linear peptide at the 3′-end may have a similar effect. Length of PMO Did Not Affect Delivery Ability of R9F2C but Affects the Antisense Activity of PMO. Tat peptide has been used to deliver various-sized cargos, ranging from small fluorescein (41) to large magnetic beads (42). The effect of cargo size on cellular delivery by an arginine-rich peptide has not been determined quantitatively. The effect of PMO length on delivery of

Moulton et al.

R9F2C conjugate was determined by flow cytometry and splice correction assay. PMO 705(12)-FL, 705-FL and 705(24)-FL are fluorescein-tagged and consist of 12, 18, and 24 bases (Table 1), respectively. R9F2C was conjugated to the 5′-end of each PMO using the GMBS linker. HeLa pLuc/705 cells treated with R9F2C-705 (12)-FL, R9F2C-705 -FL or R9F2C-705 (24)-FL exhibited identical fluorescence (Figure 8A), indicating the same amount of PMO was delivered to the cells. However, the antisense activity of the three conjugates increased with the length of PMO (Figure 8B). A longer PMO is known to have greater antisense activity than shorter ones (2), and the higher activity of longer PMO is most likely due to its higher RNA binding affinity, rather than uptake differences. To verify this, each free PMO was delivered into cells by scrape-loading, which is a mechanical method that creates transient pores in the cell membrane, allowing extracellular macromolecules to enter into the cytoplasm directly (14). The result from scrape-loading showed a similar trend as R9F2C-mediated delivery (Figure 8C) with higher antisense activity for longer PMO. Taken together, the results indicate that the length of PMO does not affect the delivery effectiveness of the R9F2C peptide but affects the antisense activity. Culture Conditions Affected the Cellular Uptake of R9F2C-PMO Conjugate. The effect of cell density, type of medium, and the serum concentration within the medium were examined by the splice correction assay. All were found to greatly influence the uptake of the conjugates. Cells treated with R9F2C-705 at cell confluence of 40-50%, 70-80%, or 90-100% produced 52, 40, or 28 RLU/µg protein, respectively (Figure 9A). Therefore, lower cell density was ideal when testing the peptidePMO conjugates. The choice of culture media was examined by adapting the HeLa pLuc705 cells in RPMI, DME/ F12, or MEM media for at least two weeks prior to treatment with the R9F2C-705 conjugate. Cells treated with the conjugate in RPMI medium had the highest luciferase production (40 RLU/µg protein) compared to those treated in DME/F12 (28 RLU/µg protein) or MEM (11 RLU/µg protein) (Figure 9B). The type and concentration of components vary among these culture media. Therefore, the difference in cellular uptake of the conjugate may be the result of differential interaction between the positively charged conjugate and components of the medium.

Figure 8. Length of PMO did not affect the delivery ability of R9F2C but affects the antisense activity. (A). Mean fluorescence of HeLa pLuc705 cells treated with 1 µM R9F2C conjugated to fluorescein-tagged PMO with 12 bases (R9F2C-705(12)-FL), 18 bases (R9F2C-705-FL), or 24 bases (R9F2C-705(24)-FL), determined by flow cytometry. The cells were treated with the test substance for 7 h in serum-free medium. (B). Luciferase activity of cells treated with the 1 µM conjugate for 7 h in serum-free RPMI medium. (C). Luciferase activity of cells treated with the 5 µM unconjugated PMO for 7 h. The unconjugated PMO was delivered by the scrapeloading method (14). Each data point is an average ( the range of means of two independent experiments and each consisting of a triplicate sample.



Figure 10. Cellular toxicity of the conjugate. Percentage viable HeLa S2(HeLa pLuc705) or HeLa cells treated with the indicated conjugate at 10 µM for 24 h, determined by MTT assay. Each data point represents the mean of a triplicate sample ( SEM.

Figure 9. Cell culture conditions affected the cellular uptake of R9F2C-PMO conjugate. Luciferase activity of HeLa pLuc705 cells treated with the test substances. Each data point represents the mean of a triplicate sample ( SEM. (A). Cells at indicated cell density treated with 10 µM R9F2C-705 for 24 h in RPMI medium with 10% FBS. (B). Cells treated with 10 µM R9F2C-705 in indicated culture medium for 24 h in 10% FBS. (C). Cells treated with 5 µM of R9F2C-705-FL or D-R9F2C-705FL for 24 h in RPMI medium containing indicated % of FBS. (D). Cells treated with 10 µM R9F2C-705 for 6 h in RPMI medium containing 1% FBS and indicated amount of additional BSA.

The effect of serum concentration in the culture medium was determined by incubating the cells with concentrations of FBS from 0% to 100%. Cells produced less luciferase as the concentration of FBS increased. Luciferase production was near background when the percentage of serum in the cultured media was >50% (Figure 9C). Positively charged molecules are known to bind to serum components. R9F2C is a highly positively charged peptide made of L-amino acids and may bind to serum components and/or be cleaved by proteinases in the serum. These two factors would decrease the peptide’s delivery effectiveness resulting in decreased antisense activity. To explore whether serum binding and/or serum proteinases were the cause of a decrease in antisense activity, a R9F2C peptide made of D-amino acids (DR9F2C) was used in a side-by-side experiment against R9F2C. D-R9F2C cannot be degraded by FBS proteinase. The result from D-R9F2C-705 conjugate was nearly identical to that from R9F2C-705 (Figure 9C), indicating that the FBS proteinases did not affect the antisense activity. This points to the binding of serum components as the main cause for reduced antisense activity of a conjugate. Bovine serum albumin (BSA) is one of the major serum proteins. Whether it inhibits cellular uptake of the conjugate was examined in the splice correction assay. The splice correction was not inhibited by addition of BSA to the medium (Figure 9D), showing BSA is not the component that inhibits the function of the conjugate. Cellular Toxicity. The conjugates of arginine-rich peptide and PMO exhibited dose-dependent toxicity (data not shown). The degree of toxicity depended on cell-type and was influenced by the number of arginines, with

greater number of arginines being more toxic (Figure 10). The difference in cellular toxicity was seen even within the subtypes of the HeLa cell line. The HeLa S2 cells (HeLa pLuc705) or HeLa cells treated with 10 µM of R9F2C-705 exhibited 83% ((1.4 SEM) or 65% ((0.61 SEM) cell viability (Figure 10). Increasing serum concentrations in the media decreases cellular toxicity of the conjugate (data not shown). The nature of this cytotoxicity is not well understood. Formation of transmembrane pores is not the cause of the toxicity because cells treated with the conjugate did not have elevated extracellular lactate dehydrogenase levels (data not shown). It is observed that adherent cells treated with the conjugate at high concentrations detached more readily from the culture wells compared to untreated cells. In summary, the conjugation of R9F2C peptide to a PMO provided a simple and efficient delivery of the PMO to the RNA target. The length of PMO did not affect the delivery efficacy of the peptide while other factors did. When using the conjugate to study gene function in cell culture, the 5′-end of PMO is the preferred end for conjugation and a flexible linker of 15 Å was the best length of these tested. We have found that R9F2C peptide delivers PMO effectively into many different cell lines; however, the cell culture conditions greatly affect the antisense activity of the conjugate. Cell density, type of medium, and serum concentration along with their relationships with cytotoxicity in the specific cell type studied are important considerations when using the conjugate of an arginine-rich peptide and an uncharged antisense oligomer to study gene function in cell culture. ACKNOWLEDGMENT

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Cellular Uptake of Antisense Morpholino Oligomers Conjugated to

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