Cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides

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. R(9)F(2)C was best suited to deliver a PMO to its target RNA resulting in the strongest antisense effect. By simply adding the R(9)F(2)C-PMO conjugate into the cell culture medium at low microM 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. R(9)F(2)C 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.     

Antiproliferative effects of steric blocking phosphorodiamidate morpholino antisense agents directed against c-myc

The phosphorodiamidate Morpholino oligomers (PMO) are a new class of antisense agents that inhibit gene expression by binding to RNA and sterically blocking processing or translation. In a search for a Morpholino agent that would inhibit cell proliferation, it was found that oligomers directed against c-myc, a gene involved in control of the cell cycle, were effective. The sequence specificity and mechanism of action of one agent were determined. The 20-mer 126 lowers c-myc protein levels in treated cells and arrests cells in G0/G1 of the cell cycle. It also acts at the RNA level to inhibit normal pre-mRNA splicing and instead produces an aberrantly spliced mRNA. Irrelevant and mispair control oligomers indicated that the observed antiproliferative effect was sequence specific. This was confirmed in a reporter gene model system using a c-myc 5'-untranslated region (5'-UTR) fused to a cDNA copy of the insect luciferase gene. We conclude that 126 is acting through an antisense mechanism involving Watson-Crick hydrogen bonding to its target RNA. A specific antisense agent directed against a cell cycle-associated gene mRNA may be useful as a therapeutic in diseases characterized by excess cell proliferation, such as restenosis following balloon angioplasty or cancer.     

Efficient splicing correction by PNA conjugation to an R6-Penetratin delivery peptide

Sequence-specific interference with the nuclear pre-mRNA splicing machinery has received increased attention as an analytical tool and for development of therapeutics. It requires sequence-specific and high affinity binding of RNaseH-incompetent DNA mimics to pre-mRNA. Peptide nucleic acids (PNA) or phosphoramidate morpholino oligonucleotides (PMO) are particularly suited as steric block oligonucleotides in this respect. However, splicing correction by PNA or PMO conjugated to cell penetrating peptides (CPP), such as Tat or Penetratin, has required high concentrations (5–10μM) of such conjugates, unless an endosomolytic agent was added to increase escape from endocytic vesicles. We have focused on the modification of existing CPPs to search for peptides able to deliver more efficiently splice correcting PNA or PMO to the nucleus in the absence of endosomolytic agents. We describe here R₆-Penetratin (in which arginine-residues were added to the N-terminus of Penetratin) as the most active of all CPPs tested so far in a splicing correction assay in which masking of a cryptic splice site allows expression of a luciferase reporter gene. Efficient and sequence-specific correction occurs at 1μM concentration of the R6Pen–PNA705 conjugate as monitored by luciferase luminescence and by RT-PCR. Some aspects of the R6Pen–PNA705 structure–function relationship have also been evaluated.     

Haloperidol-associated stealth liposomes: a potent carrier for delivering genes to human breast cancer cells

Sigma receptors are membrane-bound proteins that are overexpressed in certain human malignancies including breast cancer. These receptors show very high affinity for various sigma ligands including neuroleptics like haloperidol. We hypothesized that in associating haloperidol-linked lipid into the cationic lipid-DNA complex, we can specifically target and deliver genes to breast cancer cells that overexpress sigma receptors. In the present study, haloperidol was chemically modified to conjugate at the distal end of the polyethylene glycollinked phospholipid, which was then incorporated into the cationic liposome known to condense and deliver genes inside cells. The resulting haloperidol-conjugated targeted lipoplex showed at least 10-fold higher (p < 0.001) reporter gene expression in MCF-7 cells than control lipoplex. The reporter gene expression of the targeted lipoplex was significantly blocked by haloperidol (p < 0.001) and by another sigma ligand, 1,3-ditolylguanidine (p < 0.001) in the majority of cationic lipid to DNA charge ratios (+/-). Spironolactone-mediated sigma receptor down-regulation enabled MCF-7 to show 10-fold lower transgene expression with targeted lipoplex compared with that obtained in spironolactone-untreated cells. The targeted lipoplex generated nonspecific gene expression in sigma receptor-nonexpressing human cancer cells such as Hela, KB, HepG2, and Chinese hamster ovary cells. Moreover, the transgene expression remained unabated in physiologically relevant serum concentrations. This is the first study to demonstrate that haloperidol-targeted gene delivery systems can mediate efficient targeting of genes to sigma receptor-overexpressing breast cancer cells, thereby becoming a novel class of therapeutics for the treatment of human cancers.     

Arginine-rich peptide conjugation to morpholino oligomers: effects on antisense activity and specificity

Noncharged antisense compounds, such as phosphorodiamidate morpholino oligomers (PMOs), do not readily enter mammalian cells in culture. A simple and effective means for cellular delivery of PMOs is through their conjugation to arginine-rich peptides. Understanding the effect of peptide conjugation on the efficacy, toxicity, and specificity of PMOs is important to the successful application of this antisense delivery method. We investigated the effects of conjugation of arginine-rich peptides to PMO on the thermal stability, efficacy and specificity for targeted RNA of the resulting compound. In vitro translation assays showed that (1) R9F2-PMO generated antisense activity 3-25-fold higher than corresponding nonconjugated PMO, (2) the level of antisense activity enhancement by R9F2-PMO over a corresponding nonconjugated PMO is related to the GC content of the PMO sequence, (3) R9F2 conjugation reduced the minimum length of a PMO required to inactivate a target RNA from 20 bases to 14 bases, and (4) nonspecific effects of R9F2-PMO occur at lower concentrations than corresponding PMO alone. Thermal stability of heteroduplexes of PMO and complementary RNA were increased by conjugation of PMO to R9F2 peptide, likely accounting for the increased specific antisense activity of conjugated over nonconjugated PMO. A cell-culture based assay demonstrated that while conjugation to unnatural peptides increased PMO efficacy without causing nonspecificity at concentrations < or = 10 microM, only L-peptide conjugation retained high specificity at higher concentrations. This study demonstrates that conjugation of PMO to an arginine-rich peptide generally increases the binding affinity of the PMO to complementary RNA and increases its antisense potency. Additionally, it is shown that the enzymatic stability of an L- or unnatural peptide used for PMO conjugation affects the antisense properties of the resulting compound.     

Protamine-fragment peptides fused to an SV40 nuclear localization signal deliver oligonucleotides that produce antisense effects in prostate and bladder carcinoma cells

The development of antisense technology has focused on improving methods for oligonucleotide delivery into cells. In the present work, we describe a novel strategy for oligonucleotide delivery based on a bifunctional peptide composed of a C-terminal protamine-fragment that contains a DNA-binding domain and an N-terminal nuclear localization signal sequence derived from the SV40 large-T antigen (The sequences of two of the peptides are R6WGR6-PKKKRKV [s-protamine-NLS] and R4SR6FGR6VWR4-PKKKRKV [l-protamine-NLS]). We demonstrated, by intrinsic fluorescence quenching, that peptides of this class form complexes with oligodeoxynucleotides. To evaluate delivery, we used a 20-mer phosphorothioate oligomer (Isis 3521) targeted to the 3'-untranslated region of the PKC-alpha mRNA and G3139, an 18-mer phosphorothioate targeted to the first six codons of the human bcl-2 open reading frame, and complexed them with either of two peptides (s- or l-protamine-NLS). These peptides bind to and deliver antisense oligonucleotides to the nucleus of T24 bladder and PC3 prostate cancer cells, as demonstrated by confocal microscopy. Furthermore, as shown by Western and Northern blotting, the peptide-oligonucleotide complexes produced excellent downregulation of the expression of the complementary mRNAs, which in turn resulted in downregulation of protein expression. However, under certain circumstances (predominantly in PC3 cells), incubation of the cells with chloroquine was required to produce antisense activity. Using this strategy, PKC-alpha protein and mRNA expression in T24 and PC3 cells and bcl-2 expression in PC3 cells was reduced by approximately 75 +/- 10% at a minimum concentration of oligomer of 0.25 microM, in combination with 12-15 microM peptide. On the basis of our results, we conclude that arginine-rich peptides of this class may be potentially useful delivery vehicles for the cellular delivery of antisense oligonucleotides. This new strategy may have several advantages over other methods of oligonucleotide delivery and may complement already existing lipid-based technologies.     

Effects of base modifications on antisense properties of 2'-O-methoxyethyl and PNA oligonucleotides

A recently developed antisense splicing assay was used to determine the relative activities of 2'-O-methoxyethoxy (2'-MOE) phosphorothioate oligonucleotides containing base modifications. In the assay, RNase H-inactive oligonucleotides are used to block aberrant splicing and restore correct splicing of an Enhanced Green Fluorescence Protein (EGFP) reporter pre-mRNA stably expressed in HeLa cells. Thus, the extent of EGFP upregulation is proportional to the antisense activity of the tested molecule. The base modifications included C-5 propynyl analogs of uridine and cytidine and phenoxazine and G-clamp analogs of cytosine. Base-modified 2'-MOE oligonucleotides were delivered to the HeLa EGFP-654 test cells by cationic lipid transfection or scrape-loading or without any delivery method (free uptake). When delivered with a cationic lipid, the G-clamp and phenoxazine oligomers showed increases in activity over the unmodified 2'-MOE parent compound. However, when delivered by scrape-loading or without a delivery method, the unmodified oligomer performed best. The results suggest that base modifications do not enhance the free uptake activity of RNase H inactive 2'-MOE oligomers.     

Intracellular delivery of an anionic antisense oligonucleotide via receptor-mediated endocytosis

We describe the synthesis and characterization of a 5′ conjugate between a 2′-O-Me phosphorothioate antisense oligonucleotide and a bivalent RGD (arginine–glycine–aspartic acid) peptide that is a high-affinity ligand for the αvβ3 integrin. We used αvβ3-positive melanoma cells transfected with a reporter comprised of the firefly luciferase gene interrupted by an abnormally spliced intron. Intranuclear delivery of a specific antisense oligonucleotide (termed 623) corrects splicing and allows luciferase expression in these cells. The RGD–623 conjugate or a cationic lipid-623 complex produced significant increases in luciferase expression, while ‘free’ 623 did not. However, the kinetics of luciferase expression was distinct; the RGD–623 conjugate produced a gradual increase followed by a gradual decline, while the cationic lipid-623 complex caused a rapid increase followed by a monotonic decline. The subcellular distribution of the oligonucleotide delivered using cationic lipids included both cytoplasmic vesicles and the nucleus, while the RGD–623 conjugate was primarily found in cytoplasmic vesicles that partially co-localized with a marker for caveolae. Both the cellular uptake and the biological effect of the RGD–623 conjugate were blocked by excess RGD peptide. These observations suggest that the bivalent RGD peptide–oligonucleotide conjugate enters cells via a process of receptor-mediated endocytosis mediated by the αvβ3 integrin.     

Bi-specific splice-switching PMO oligonucleotides conjugated via a single peptide active in a mouse model of Duchenne muscular dystrophy

The potential for therapeutic application of splice-switching oligonucleotides (SSOs) to modulate pre-mRNA splicing is increasingly evident in a number of diseases. However, the primary drawback of this approach is poor cell and in vivo oligonucleotide uptake efficacy. Biological activities can be significantly enhanced through the use of synthetically conjugated cationic cell penetrating peptides (CPPs). Studies to date have focused on the delivery of a single SSO conjugated to a CPP, but here we describe the conjugation of two phosphorodiamidate morpholino oligonucleotide (PMO) SSOs to a single CPP for simultaneous delivery and pre-mRNA targeting of two separate genes, exon 23 of the Dmd gene and exon 5 of the Acvr2b gene, in a mouse model of Duchenne muscular dystrophy. Conjugations of PMOs to a single CPP were carried out through an amide bond in one case and through a triazole linkage (‘click chemistry’) in the other. The most active bi-specific CPP–PMOs demonstrated comparable exon skipping levels for both pre-mRNA targets when compared to individual CPP–PMO conjugates both in cell culture and in vivo in the mdx mouse model. Thus, two SSOs with different target sequences conjugated to a single CPP are biologically effective and potentially suitable for future therapeutic exploitation.     

Inhibition of cell adhesion to plastic substratum by phosphorothioate oligonucleotide.

Antisense oligonucleotides have been widely used to achieve specific inhibition of targeted gene expression. However, the mechanism of action is not well understood and in many systems sequence-independent effects occur. We have recently shown that chronic administration of an antisense c-myc phosphorothioate oligonucleotide can specifically inhibit expression of the c-myc protein and growth in human breast cancer cells. We now identify an additional effect of the same oligonucleotide on cell adhesion. Transient delivery through electroporation of 2.5 microM antisense-myc oligonucleotide to MCF-7 cells results in 85% inhibition of adhesion to plastic substratum within 24 h. Both the onset of this effect and the subsequent recovery occur without a change in cell viability, growth, or alteration of adhesion to Matrigel, collagen IV, laminin, or fibronectin. However, no parallel changes in c-myc mRNA or protein expression are detectable, suggesting that in this instance inhibition of adhesion caused by antisense-myc oligonucleotide may involve a mechanism independent of the target sequence.     

Enhanced exon skipping and prolonged dystrophin restoration achieved by TfR1-targeted delivery of antisense oligonucleotide using FORCE conjugation in mdx mice

Current therapies for Duchenne muscular dystrophy (DMD) use phosphorodiamidate morpholino oligomers (PMO) to induce exon skipping in the dystrophin pre-mRNA, enabling the translation of a shortened but functional dystrophin protein. This strategy has been hampered by insufficient delivery of PMO to cardiac and skeletal muscle. To overcome these limitations, we developed the FORCE^TM platform consisting of an antigen-binding fragment, which binds the transferrin receptor 1, conjugated to an oligonucleotide. We demonstrate that a single dose of the mouse-specific FORCE–M23D conjugate enhances muscle delivery of exon skipping PMO (M23D) in mdx mice, achieving dose-dependent and robust exon skipping and durable dystrophin restoration. FORCE–M23D-induced dystrophin expression reached peaks of 51%, 72%, 62%, 90% and 77%, of wild-type levels in quadriceps, tibialis anterior, gastrocnemius, diaphragm, and heart, respectively, with a single 30 mg/kg PMO-equivalent dose. The shortened dystrophin localized to the sarcolemma, indicating expression of a functional protein. Conversely, a single 30 mg/kg dose of unconjugated M23D displayed poor muscle delivery resulting in marginal levels of exon skipping and dystrophin expression. Importantly, FORCE–M23D treatment resulted in improved functional outcomes compared with administration of unconjugated M23D. Our results suggest that FORCE conjugates are a potentially effective approach for the treatment of DMD.     

Delivery of antisense oligonucleotides and plasmid DNA with various carrier agents

A series of cationic nucleic acid carriers was evaluated for their ability to deliver pLuc plasmid DNA or a 2'-O-methyl-oligoribonucleoside phosphorothioate, ON-705. Oligonucleotide delivery and its antisense function were assayed by a recently developed assay based on alternative splicing of modified luciferase pre-mRNA (Kang et al., 1998). This assay scores only the nuclear and sequence-specific antisense activity of the oligonucleotides. The results show that the efficiencies of delivery of plasmid DNA and oligonucleotides by the tested carriers, with the exception of Exgene and Lipofectamine, differed markedly. The efficiency of the delivery of ON-705 oligonucleotide was reduced by 70%-90% for all carriers, except Effectene, in culture media containing 8% fetal bovine serum. Interestingly, the efficiency of delivery of the ON-705-Effectene complex increased with serum concentrations of up to 30%.     

Novel cationic amphiphiles as delivery agents for antisense oligonucleotides.

There has been great interest recently in therapeutic use of nucleic acids including genes, ribozymes and antisense oligonucleotides. Despite recent improvements in delivering antisense oligonucleotides to cells in culture, nucleic acid-based therapy is still often limited by the poor penetration of the nucleic acid into the cytoplasm and nucleus of cells. In this report we describe nucleic acid delivery to cells using a series of novel cationic amphiphiles containing cholic acid moieties linked via alkylamino side chains. We term these agents 'molecular umbrellas' since the cationic alkylamino chains provide a 'handle' for binding of nucleic acids, while the cholic acid moieties are likely to interact with the lipid bilayer allowing the highly charged nucleic acid backbone to traverse across the cell membrane. Optimal gene and oligonucleotide delivery to cells was afforded by a derivative (amphiphile 5) containing four cholic acid moieties. With this amphiphile used as a constituent in cationic liposomes, a 4-5 log increase in reporter gene delivery was measured. This amphiphile used alone provided a 250-fold enhancement of oligo-nucleotide association with cells as observed by flow cytometry. A substantial fraction of cells exposed to complexes of amphiphile 5 and fluorescent oligo-nucleotide showed nuclear accumulation of the fluorophore. Enhanced pharmacological effectiveness of antisense oligonucleotides complexed with amphiphile 5 was observed using an antisense splicing correction assay that activates a Luciferase reporter. Intracellular delivery, nuclear localization and pharmacological effectiveness of oligonucleotides using amphiphile 5 were similar to those afforded by commercial cytofectins. However, in contrast to most commercial cytofectins, the umbrella amphiphile showed substantial delivery activity even in the presence of high concentrations of serum.     

Modification of alternative splicing of Bcl-x pre-mRNA in prostate and breast cancer cells. analysis of apoptosis and cell death

There is ample evidence that deregulation of apoptosis results in the development, progression, and/or maintenance of cancer. Since many apoptotic regulatory genes (e.g. bcl-x) code for alternatively spliced protein variants with opposing functions, the manipulation of alternative splicing presents a unique way of regulating the apoptotic response. Here we have targeted oligonucleotides antisense to the 5'-splice site of bcl-x(L), an anti-apoptotic gene that is overexpressed in various cancers, and shifted the splicing pattern of Bcl-x pre-mRNA from Bcl-x(L) to Bcl-x(S), a pro-apoptotic splice variant. This approach induced significant apoptosis in PC-3 prostate cancer cells. In contrast, the same oligonucleotide treatment elicited a much weaker apoptotic response in MCF-7 breast cancer cells. Moreover, although the shift in Bcl-x pre-mRNA splicing inhibited colony formation in both cell lines, this effect was much less pronounced in MCF-7 cells. These differences in responses to oligonucleotide treatment were analyzed in the context of expression of Bcl-x(L), Bcl-x(S), and Bcl-2 proteins. The results indicate that despite the presence of Bcl-x pre-mRNA in a number of cell types, the effects of modification of its splicing by antisense oligonucleotides vary depending on the expression profile of the treated cells.     

Structural requirements for cellular uptake and antisense activity of peptide nucleic acids conjugated with various peptides

Peptide nucleic acids (PNAs) have shown great promise as potential antisense drugs; however, poor cellular delivery limits their applications. Improved delivery into mammalian cells and enhanced biological activity of PNAs have been achieved by coupling to cell-penetrating peptides (CPPs). Structural requirements for the shuttling ability of these peptides as well as structural properties of the conjugates such as the linker type and peptide position remained controversial, so far. In the present study an 18mer PNA targeted to the cryptic splice site of a mutated beta-globin intron 2, which had been inserted into a luciferase reporter gene coding sequence, was coupled to various peptides. As the peptide lead we used the cell-penetrating alpha-helical amphipathic peptide KLAL KLAL KAL KAAL KLA-NH2 [model amphipathic peptide (MAP)] which was varied with respect to charge and structure-forming properties. Furthermore, the linkage and the localization of the attached peptide (C- vs N-terminal) were modified. Positive charge as well as helicity and amphipathicity of the KLA peptide was all required for efficient dose-dependent correction of aberrant splicing. The highest antisense effect was reached within 4 h without any transfection agent. Stably linked conjugates were also efficient in correction of aberrant splicing, suggesting that a cleavable disulfide bond between CPP and PNA is clearly not essential. Moreover, the placement of the attached peptide turned out to be crucial for attaining antisense activity. Coadministration of endosome disrupting agents such as chloroquine or Ca2+ significantly increased the splicing correction efficiency of some conjugates, indicating the predominant portion to be sequestered in vesicular compartments.     

Enhanced delivery of antisense oligonucleotides with fluorophore-conjugated PAMAM dendrimers

PAMAM dendrimers are cationic polymers that have been used for the delivery of genes and oligonucleotides to cells. However, little is known about the behavior of dendrimer–nucleic acid complexes once they reach the cell interior. To pursue this issue, we prepared dendrimers conjugated with the fluorescent dye Oregon green 488. These were used in conjunction with oligonucleotides labeled with a red (TAMRA) fluorophore in order to visualize the sub-cellular distribution of the dendrimer–oligonucleotide complex and of its components by two-color digital fluorescence microscopy. The 2′-O-methyl antisense oligonucleotide sequence used in these studies was designed to correct splicing at an aberrant intron inserted into a luciferase reporter gene; thus effective delivery of the antisense agent results in the expression of the reporter gene product. The dendrimer–oligonucleotide complex remained associated during the process of uptake into vesicular compartments and eventual entry into the nucleus. Since the pharmacological activity of the antisense compound was manifest under these conditions, it suggests that the dendrimer–oligonucleotide complex is functionally active. A surprising result of these studies was that the Oregon green 488-conjugated dendrimer was a much better delivery agent for antisense compounds than unmodified dendrimer. This suggests that coupling of relatively hydrophobic small molecules to PAMAM dendrimers may provide a useful means of enhancing their capabilities as delivery agents for nucleic acids.     

Subnanomolar antisense activity of phosphonate-peptide nucleic acid (PNA) conjugates delivered by cationic lipids to HeLa cells

In the search of facile and efficient methods for cellular delivery of peptide nucleic acids (PNA), we have synthesized PNAs conjugated to oligophosphonates via phosphonate glutamine and bis-phosphonate lysine amino acid derivatives thereby introducing up to twelve phosphonate moieties into a PNA oligomer. This modification of the PNA does not interfere with the nucleic acid target binding affinity based on thermal stability of the PNA/RNA duplexes. When delivered to cultured HeLa pLuc705 cells by Lipofectamine, the PNAs showed dose-dependent nuclear antisense activity in the nanomolar range as inferred from induced luciferase activity as a consequence of pre-mRNA splicing correction by the antisense-PNA. Antisense activity depended on the number of phosphonate moieties and the most potent hexa-bis-phosphonate-PNA showed at least 20-fold higher activity than that of an optimized PNA/DNA hetero-duplex. These results indicate that conjugation of phosphonate moieties to the PNA can dramatically improve cellular delivery mediated by cationic lipids without affecting on the binding affinity and sequence discrimination ability, exhibiting EC(50) values down to one nanomolar. Thus the intracellular efficacy of PNA oligomers rival that of siRNA and the results therefore emphasize that provided sufficient in vivo bioavailability of PNA can be achieved these molecules may be developed into potent gene therapeutic drugs.