Specific Delivery of Oligonucleotides to the Cell Nucleus via Gentle Compression and Attachment of Polythymidine.

Nonviral delivery of nucleic acids to the cell nucleus typically requires chemical methods that do not guarantee specific delivery (e.g., transfection agent) or physical methods that may require extensive fabrication (e.g., microfluidics) or an elevated pressure (e.g., 105 Pa for microneedles). We report a method of delivering oligonucleotides to the nucleus with high specificity (relative to the cytosol) by synergistically combining chemical and physical approaches. Particularly, we demonstrate that DNA oligonucleotides appended with a polythymidine [poly(T)] segment (chemical) profusely accumulate inside the nucleus when the cells are under gentle compression imposed by the weight of a single glass coverslip (physical; ∼2.2 Pa). Our "compression-cum-poly(T)" delivery method is simple, can be generalizable to three "hard-to-transfect" cell types, and does not induce significant levels of cytotoxicity or long-term oxidative stress to the treated cells when provided the use of suitable compression times and oligonucleotide concentrations. In bEnd.3 endothelial cells, compression-aided intranuclear delivery of poly(T) is primarily mediated by importin ß and nucleoporin 62. Our method significantly enhances the intranuclear delivery of antisense oligonucleotides to bEnd.3 endothelioma cells and the inhibition of two target genes, including a reporter gene encoding the enhanced green fluorescent protein and an intranuclear lncRNA oncogene (metastasis-associated lung adenocarcinoma transcript 1), when compared with delivery without gentle compression or poly(T) attachment. Our data underscore the critical roles of pressure and nucleotide sequence on the intranuclear delivery of nucleic acids.

Orally Delivered Antisense Oligodeoxyribonucleotides of TNF-α via Polysaccharide-Based Nanocomposites Targeting Intestinal Inflammation.

Tumor necrosis factor alpha (TNF-α) is usually regarded as a potential target for inflammatory bowel disease therapy. Herein, a promising strategy for effective delivery of phosphorothioated antisense oligodeoxyribonucleotide of TNF-α (PS-ATNF-α), targeting the intestinal inflammation based on the interaction of the single chain of triple helical ß-glucan (s-LNT) with poly-deoxyadenylic acid [poly(dA)], and the colon-specific degradation of chitosan-alginate (CA) hydrogel, is reported. The target gene of PS-ATNF-α, with a poly(dA) tail through a disulfide bond (-SS-), interacts with s-LNT to form a rod-like nanocomposite of s-LNT/poly(dA)-SS-PS-ATNF-α, which significantly inhibits lipopolysaccharide (LPS)-induced TNF-α at the protein level by 38.2% and mRNA level by 48.9% in RAW264.7 macrophages. The nanocomposites carried by the CA hydrogel with the loading amount of 83.5% are then orally administered and specifically released to the inflamed intestine, followed by internalization into intestinal cells such as macrophages, to reduce TNF-α production by 36.4% and dextran sulfate sodium-induced inflammation by decreasing myeloperoxidase and malondialdehyde. This study defines a new strategy for the oral delivery of antisense oligonucleotides to attenuate inflammatory response, demonstrating a notable potential for clinical applications in intestine-inflammation-targeted therapy.

Intracellular oligonucleotide delivery using the cell penetrating peptide Xentry.

The current study investigated the use of two cationic peptides, Xentry-KALA (XK) and Xentry-Protamine (XP), for intracellular delivery of Connexin43 antisense oligonucleotides (Cx43AsODN). The charge and size of Cx43AsODN:XK and Cx43AsODN:XP complexes was determined by Zetasizer analysis. The earliest positive zeta potential reading was obtained at a 1:2 and 1:1.2 charge ratio of Cx43AsODN:XK and Cx43AsODN:XP respectively, with Cx43AsODN:XK resulting in overall larger complexes than Cx43AsODN:XP. Gel shift mobility assays revealed complete complex formation at a 1:2.5 and 1:2.2 charge ratio of Cx43AsODN:XK and Cx43AsODN:XP, respectively. Cellular uptake studies were carried out in ARPE-19 cells. While both complexes were able to enter the cells, Cx43AsODN:XK uptake appeared punctate and circular indicative of endosomal containment. Cx43AsODN:XP uptake, in contrast, resulted in diffuse appearance inside the cell suggesting endosomal escape of the cargo. Finally, western blot analysis confirmed that Cx43AsODN:XP was able to knockdown Cx43 expression in these cells under normal and hypoxic conditions.

Quantitative relationship between chemical properties and bioactivities of anti-microRNA oligonucleotides targeted to tumor-associated microRNA-21.

Synthetic anti-microRNA oligonucleotides (AMOs) are promising drug candidates to inactivate disease-related microRNAs because of their sequence-specific binding to their targets and the variety of chemical modifications available. Over the last decade, the qualitative relationships between the chemical properties of AMOs and bioactivity (inactivation of their target miRNAs) have been studied to enhance their bioactivity. On the other hand, in real-world drug development, drugs must be designed case-by-case, taking many factors into account. Thus, in order to design AMOs that target specific miRNA, understanding the quantitative relationship between the chemical properties of AMOs and inactivation of their target miRNA is necessary. Here, we aimed to find the specific quantitative relationship of AMOs targeted to tumor-associated miR-21 through direct comparison of their inactivation efficacies with systematically varied chemical properties, including sequence-specific binding affinity, nuclease resistance, and RNase H activation. As a result, we newly found the quantitative relationships; (1) sequence-specific binding affinity of AMOs against miR-21 is the main determining factor for inactivation efficacy, (2) nuclease resistance of AMOs impacts their miR-21 inactivation efficacy acting cooperatively with the binding affinity, although nuclease resistance alone does not affect the miRNA inactivation efficacy, and (3) RNase H activation is unnecessary. This study also demonstrates the utility of the obtained relationship for the design of AMO-based drugs targeted to miR-21, through cell-based analyses. Thus, the obtained quantitative relationship would make it possible to predict the miR-21 inactivation efficacy of AMOs which are newly designed.

Cross-Linking Antisense Oligodeoxyribonucleotides with a Photoresponsive α-Chloroaldehyde Moiety for RNA Point Mutations.

Because point mutations in GTPase-coding genes have been reported to be responsible for the transformation of cells, anticancer reagents that react effectively and sequence selectively with target RNAs having a point mutation are highly desired. In this study, we developed novel photo-cross-linking oligodeoxyribonucleotides ((pro)PCA-ODNs) that had a caged α-chloroaldehyde group conjugated to a 2-methylpropanediyl backbone ((pro)PCA) in the middle of the strand. A kinetic study of the deprotection reaction of (pro)PCA-ODN revealed that the bis(2-nitrobenzyl)acetal group was completely deprotected within 1 min. Photo-cross-linking studies of (pro)PCA-ODNs with complementary oligoribonucleotides (ORNs) revealed that (pro)PCA-ODNs reacts efficiently and selectively with the target ORNs that have an adenosine or cytidine residue at a frontal position of the (pro)PCA residue without adverse effects of bases adjacent to the mutation site.

Inhibition of choriocarcinoma by Fe3O4-dextran-anti-ß-human chorionic gonadotropin nanoparticles containing antisense oligodeoxynucleotide of heparanase.

OBJECTIVE: To observe the influence of Fe3O4-dextran-anti-ß-human chorionic gonadotropin (HCG) carrying heparanase (Hpa) antisense oligodeoxynucleotide (ASODN), via the invasion, proliferation, and Hpa expression of JEG-3 cell lines and inhibitory effect of transplanted choriocarcinoma tumor growth. METHODS: The different abilities of invasion and proliferation between transfected JEG-3 and untransfected JEG-3 were measured by Matrigel invasion assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in vitro. The effect of Hpa ASODN transfection on the expression of Hpa mRNA and protein was measured by reverse-transcription polymerase chain reaction and Western blot. The transplanted choriocarcinoma tumors were taken out to calculate the inhibitory effect on tumor growth of Hpa ASODN. RESULTS: IN THIS STUDY, WE FOUND THAT: (1) the invasive ability of JEG-3 cells was inhibited sufficiently (P < 0.05) after JEG-3 cells were transfected by Fe3O4-dextran-anti-ßHCG carrying Hpa ASODN; (2) after JEG-3 cells were transfected by Fe3O4-dextran-anti-ßHCG carrying Hpa ASODN at 48 and 72 hours, the proliferative ability of JEG-3 cells was inhibited sufficiently (P < 0.05); (3) the expression of Hpa mRNA and protein in JEG-3 cells was inhibited efficiently after JEG-3 cells were transfected by Fe3O4-dextran-anti-ßHCG carrying Hpa ASODN (P < 0.05); and (4) Fe3O4-dextran-anti-ßHCG carrying Hpa ASODN had an inhibitory effect on the transplanted choriocarcinoma tumor growth (P < 0.05) and was harmless on nude mice. CONCLUSION: Fe3O4-dextran-anti-ßHCG carrying Hpa ASODN weakened the invasive and proliferative ability of choriocarcinoma, with a significant inhibitory effect on the transplanted choriocarcinoma tumor. Therefore, Fe3O4-dextran-anti-ßHCG carrying Hpa ASODN is an effective gene therapy, and Fe3O4-dextran-anti-ßHCG nanoparticles are a harmless and effective gene vector.

Structure-transfection activity relationships in a series of novel cationic lipids with heterocyclic head-groups.

Cationic liposomes are promising candidates for the delivery of various therapeutic nucleic acids. Here, we report a convenient synthesis of carbamate-type cationic lipids with various hydrophobic domains (tetradecanol, dialkylglycerol, cholesterol) and positively charged head-groups (pyridinium, N-methylimidazolium, N-methylmorpholinium) and data on the structure-transfection activity relationships. It was found that single-chain lipids possess high surface activity, which correlates with high cytotoxicity due to their ability to disrupt the cellular membrane by combined hydrophobic and electrostatic interactions. Liposomes containing these lipids also display high cytotoxicity with respect to all cell lines. Irrespective of chemical structures, all cationic lipids form liposomes with similar sizes and surface potentials. The characteristics of complexes composed of cationic liposomes and nucleic acids depend mostly on the type of nucleic acid and P/N ratios. In the case of oligodeoxyribonucleotide delivery, the transfection activity depends on the type of cationic head-group regardless of the type of hydrophobic domain: all types of cationic liposomes mediate efficient oligonucleotide transfer into 80-90% of the eukaryotic cells, and liposomes based on lipids with N-methylmorpholinium cationic head-group display the highest transfection activity. In the case of plasmid DNA and siRNA, the type of hydrophobic domain determines the transfection activity: liposomes composed of cholesterol-based lipids were the most efficient in DNA transfer, while liposomes containing glycerol-based lipids exhibited reasonable activity in siRNA delivery under serum-free conditions.

Static micromixer-coaxial electrospray synthesis of theranostic lipoplexes.

Theranostic lipoplexes are an integrated nanotherapeutic system with diagnostic imaging capability and therapeutic functions. They hold great promise to improve current cancer treatments; however, producing uniform theranostic lipoplexes with multiple components in a reproducible manner is a highly challenging task. Conventional methods, such as bulk mixing, are not able to achieve this goal because of their macroscale and random nature. Here we report a novel technique, called the static micromixer-coaxial electrospray (MCE), to synthesize theranostic lipoplexes in a single step with high reproducibility. In this work, quantum dots (QD605) and Cy5-labeled antisense oligodeoxynucleotides (Cy5-G3139) were chosen as the model imaging reagent and therapeutic drug, respectively. Compared with bulk mixing, QD605/Cy5-G3139-loaded lipoplexes produced by MCE were highly uniform with polydispersity of 0.024 ± 0.006 and mean diameter by volume of 194 ± 15 nm. MCE also showed higher encapsulation efficiency of QD605 and Cy5-G3139. QD605 and Cy5 also formed the Förster resonance energy transfer pair, and thus the cellular uptake and intracellular fate of theranostic lipoplexes could be visualized by flow cytometry and confocal microscopy. The lipoplexes were efficiently delivered to A549 cells (non-small cell lung cancer cell line) and down-regulated the Bcl-2 gene expression by 48 ± 6%.

Thiolated chitosan nanoparticles as a delivery system for antisense therapy: evaluation against EGFR in T47D breast cancer cells.

Thiolated chitosan has high transfection and mucoadhesive properties. We investigated the potential of two recently synthesized polymers: NAC-C (N-acetyl cysteine-chitosan) and NAP-C (N-acetyl penicillamine-chitosan) in anticancer drug delivery targeting epidermal growth factor receptor (EGFR). Doxorubicin (DOX) and antisense oligonucleotide (ASOND)-loaded polymer nanoparticles were prepared in water by a gelation process. Particle characterization, drug loading, and drug release were evaluated. To verify drug delivery efficiency in vitro experiments on a breast cancer cell line (T47D) were performed. EGFR gene and protein expression was analyzed by real time quantitative polymerase chain reaction and Western blotting, respectively. A loading percentage of 63% ± 5% for ASOND and 70% ± 5% for DOX was achieved. Drug release data after 15 hours showed that ASOND and DOX were completely released from chitosan-based particles while a lower and more sustained release of only 22% ± 8% was measured for thiolated particles. In a cytosol simulated release medium/reducing environment, such as found intracellularly, polymer-based nanoparticles dissociated, liberating approximately 50% of both active substances within 7 hours. ASOND-loaded polymer nanoparticles had higher stability and high mucoadhesive properties. The ASOND-loaded thiolated particles significantly suppressed EGFR gene expression in T47D cells compared with ASOND-loaded chitosan particles and downregulated EGFR protein expression in cells. This study could facilitate future investigations into the functionality of NAP-C and NAC-C polymers as an efficient ASOND delivery system in vitro and in vivo.

Uptake and intracellular fate of multifunctional nanoparticles: a comparison between lipoplexes and polyplexes via quantum dot mediated Förster resonance energy transfer.

Lipoplexes and polyplexes represent the two major nanocarrier systems for nucleic acid delivery. Previous studies examining their uptake and intracellular unpacking rely on organic fluorophores fraught with low signal intensity and photobleaching. In this work quantum dot mediated Förster resonance energy transfer (QD-FRET) was first used to study and compare the cellular uptake and the intracellular fate of oligodeoxynucelotide (ODN)-based lipoplexes and polyplexes. QD605-amine and Cy5-labeled ODN (Cy5-GTI2040) were chosen as the FRET pair. By adjusting the lipid/ODN ratio of lipoplexes and the nitrogen/phosphate (N/P) ratio of polyplexes, lipoplexes and polyplexes with comparable physical properties were produced. The biological activities of dual-labeled lipoplexes and polyplexes remained unaltered compared to their unlabeled counterparts as evidenced by their comparable antisense activities against protein R2 in KB cells. Flow cytometry and confocal microscopy revealed similar pattern of uptake for these two types of nanoparticles, although polyplexes had a higher dissociation rate than lipoplexes in KB cells. We demonstrate that QD-FRET is a sensitive tool to study the uptake and intracellular unpacking of lipoplexes and polyplexes, which may help optimize their formulations for various theranostics applications.

Therapeutic targeting of TLR9 inhibits cell growth and induces apoptosis in neuroblastoma.

The Toll-like receptor 9 (TLR9) evolved to cope with pathogens, but it is expressed in a variety of tumors for reasons that are unclear. In this study, we report that neuroblastoma (NB) cells express functional TLR9. Liposome-complexed CpG oligonucleotides inhibited the proliferation of TLR9-expressing NB cells and induced caspase-dependent apoptotic cell death. Inhibitory oligonucleotides (iODNs) abrogated these effects. RNA interference reduced TLR9 expression but not to the level where functional responses to CpG were abolished. Compared with free CpG, liposomal formulations of NB-targeted CpG (TL-CpG) significantly prolonged the survival of mice bearing NB tumor xenografts. While CpG alone lacked antitumor efficacy in NOD/SCID/IL2rg(-/-) mice, TL-CpG retained significant efficacy related to direct effects on tumor cells. TLR9 expression in primary human NB specimens was found to correlate inversely with disease stage. Our findings establish functional expression of TLR9 in NB and suggest that TLR9 may represent a novel theranostic target in this disease.

Disulfide-linked liposomes: effective delivery vehicle for Bcl-2 antisense oligodeoxyribonucleotide G3139.

BACKGROUND: Disulfide-linked oligodeoxyribonucleotide (ODN) liposomes were formulated and evaluated for the delivery of antisense ODN G3139 in KB human oral carcinoma cells. MATERIALS AND METHODS: Liposomes composed of 1,2-di-(9Z-octadecenoyl)-3-trimethylammo-nium-propane (DOTAP)/egg phosphatidylcholine/alpha-tocopheryl polyethylene glycol 1000 succinate were incorporated with hydrophobized disulfide-linked ODN. Disulfide-linked ODN liposomes were characterized for their size, ODN intracellular delivery, Bcl-2 mRNA and protein expression, growth inhibition, and chemosensitization. RESULTS: Intracellular delivery of ODN with disulfide-linked ODN liposomes was more efficient than that with non-liposomal hydrophobized disulfide-linked ODN. Treatment of the cells with disulfide-linked ODN liposomes resulted in efficient Bcl-2 down-regulation greater than that with hydrophobized disulfide-linked ODN and consistent with that of cellular growth inhibition and the sensitization to daunorubicin in KB cells. Disulfide-linked ODN liposomes exhibited superior colloidal stability during 5-week storage. CONCLUSION: Disulfide-linked liposomes are effective delivery vehicles for antisense ODN.

Delivery of antisense oligodeoxyribonucleotide lipopolyplex nanoparticles assembled by microfluidic hydrodynamic focusing.

A multi-inlet microfluidic hydrodynamic focusing (MF) system to prepare lipopolyplex (LP) containing Bcl-2 antisense deoxyoligonucleotide (ODN) was developed and evaluated. The lipopolyplex nanoparticles consist of ODN:protamine:lipids (1:0.3:12.5wt/wt ratio) and the lipids included DC-Chol:egg PC:PEG-DSPE (40:58:2mol/mol%). Using K562 human erythroleukemia cells, which contain an abundance of Bcl-2 and overexpression of transferrin receptors (TfR), and G3139 (oblimerson sodium or Genasense(TM)) as a model cell line and drug, respectively, the Bcl-2 down-regulation at the mRNA and protein levels as well as cellular uptake and apoptosis was compared between the conventional bulk mixing (BM) method and the MF method. The lipopolyplex size and surface charge were characterized by dynamic light scattering (DLS) and zeta potential (zeta) measurement, respectively, while the ODN encapsulation efficiency was determined by gel electrophoresis. Cryogenic transmission electron microscopy (Cryo-TEM) was used to determine the morphology of LPs. Our results demonstrated that MF produced LP nanoparticles had similar structures but smaller size and size distribution compared to BM LP nanoparticles. MF LP nanoparticles had higher level of Bcl-2 antisense uptake and showed more efficient down-regulation of Bcl-2 protein level than BM LP nanoparticles.

[Preparation of ASODN-protamine-HSA-PLGA nanoparticles and initial evaluation of their nucleus targeting property in vitro].

OBJECTIVES: To develop a novel non-viral gene delivery system-SODN-Protamine-HSA-PLGA (ASODN-P/H-PLGA-NP) and investigate its nucleus targeting potential in vitro. METHODS: ASODN-P/H-PLGA-NP was prepared by mixing the protamine sulfate and HSA. Then the PLGA nanoparticles were prepared using double-emulsion evaporation technique, followed by addition of ASODN to the prepared P/H complex. The morphology of ASODN-P/H-PLGA-NP was observed by transmission electron microscopy. The diameter, PDI, and surface charge of ASODN-P/H-PLGA-NP were measured by photo correlation spectroscopy (PCS). The encapsulation efficiency of ASODN was determined by double step method. The cytotoxicity of ASODN-P/H-PLGA-NP was investigated by MTT assays. The ability to enter the squamouse carcinoma: Hep-2 cell line and its nucleus targeting property were observed by confocal laser scanning microscope. RESULTS: The average diameter, PDI, zeta potential, and encapsulation efficiency of ASODN-P/H-PLGA-NP were 128 nm, 0.234, -23.3 mV, and 78.45%, respectively. ASODN-P/H-PLGA-NP could protect the ASODN from the shear force in the ultrasound process during preparation. ASODN-P/H-PLGA-NP couldenter Hep-2 cells and have certain level of nucleus targeting property. CONCLUSION: ASODN-P/H-PLGA-NP can be prepared easily with small particle sizes and low cytotoxicity, which might be employed as a good non-viral vector for applications in ASODN delivery to nucleus.

The efficiency of CD40 down regulation by siRNA and antisense ODN: comparison of lipofectamine and FuGENE6.

BACKGROUND: Dendritic cells (DCs) are ideal accessory cells in the field of gene therapy. Delivery of DNA and siRNA into mammalian cells is a useful technique in treating various diseases caused by single gene defects. Selective gene silencing by small interfering RNAs (siRNAs) and antisense oligodeoxynucleotides (ODN)s is an efficient method for the manipulation of cellular functions. An efficient, functional delivery system with no toxicity problems would be attractive. OBJECTIVE: We compared two commercially available cationic lipids, Lipofectamine and FuGENE6, in the delivery of both siRNA and antisense ODNs into mice spleen-derived DCs. METHODS: Cellular uptake was measured by the means of fluorescein-labelled non-silencing siRNA and antisense ODNs as a model system using flow cytometry. Cytotoxicity of the two delivery systems was compared with propidium iodide and annexin-V staining, and quantified with flow cytometry. The efficiency of our oligonucleotide delivery systems was compared by measuring CD40 expression by flow cytometry. RESULTS: CD40 expression in DCs was 38%. After siRNA transfection by Lipofectamine, CD40 expression decreased to 13%, and after transfection by FuGENE6, it decreased to 18%. The difference was statistically significant. CD40 down regulation in DCs transfected with the two different antisense sequences by Lipofectamine was 21% and 23%, and down regulation after transfection by FuGENE6 was 19% and 18%, respectively. The differences were not statistically significant. The effects of siRNA and antisense ODNs were specific. CONCLUSION: Lipofectamine was a more potent delivery system in siRNA effect, followed by FuGENE6. There was no significant difference between Lipofectamine and FuGENE6 as a delivery system of antisense ODNs.

Antisense therapeutics for neurofibromatosis type 1 caused by deep intronic mutations.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder affecting 1:3,500 individuals. Disease expression is highly variable and complications are diverse. However, currently there is no specific treatment for the disease. NF1 is caused by mutations in the NF1 gene, approximately 2.1% of constitutional mutations identified in our population are deep intronic mutations producing the insertion of a cryptic exon into the mature mRNA. We used antisense morpholino oligomers (AMOs) to restore normal splicing in primary fibroblast and lymphocyte cell lines derived from six NF1 patients bearing three deep intronic mutations in the NF1 gene (c.288+2025T>G, c.5749+332A>G, and c.7908-321C>G). AMOs were designed to target the newly created 5' splice sites to prevent the incorporation of cryptic exons. Our results demonstrate that AMO treatment effectively restored normal NF1 splicing at the mRNA level for the three mutations studied in the different cell lines analyzed. We also found that AMOs had a rapid effect that lasted for several days, acting in a sequence-specific manner and interfering with the splicing mechanism. Finally, to test whether the correction of aberrant NF1 splicing also restored neurofibromin function to wild-type levels, we measured the amount of Ras-GTP after AMO treatment in primary fibroblasts. The results clearly show an AMO-dependent decrease in Ras-GTP levels, which is consistent with the restoration of neurofibromin function. To our knowledge this is the first time that an antisense technique has been used successfully to correct NF1 mutations opening the possibility of a therapeutic strategy for this type of mutation not only for NF1 but for other genetic disorders.

Transferrin receptor-targeted lipid nanoparticles for delivery of an antisense oligodeoxyribonucleotide against Bcl-2.

Antisense oligonucleotide G3139-mediated down-regulation of Bcl-2 is a potential strategy for overcoming chemoresistance in leukemia. However, the limited efficacy shown in recent clinical trials calls attention to the need for further development of novel and more efficient delivery systems. In order to address this issue, transferrin receptor (TfR)-targeted, protamine-containing lipid nanoparticles (Tf-LNs) were synthesized as delivery vehicles for G3139. The LNs were produced by an ethanol dilution method, and lipid-conjugated Tf ligand was then incorporated by a postinsertion method. The resulting Tf-LNs had a mean particle diameter of approximately 90 nm and G3139 loading efficiency of 90.4%. Antisense delivery efficiency of Tf-LNs was evaluated in K562, MV4-11, and Raji leukemia cell lines. The results showed that Tf-LNs were more effective than nontargeted LNs and free G3139 (p < 0.05) in decreasing Bcl-2 expression (by up to 62% at the mRNA level in K562 cells) and in inducing caspase-dependent apoptosis. In addition, Bcl-2 down-regulation and apoptosis induced by Tf-LN G3139 were shown to be blocked by excess free Tf and thus were TfR-dependent. Cell lines with higher TfR expression also showed greater Bcl-2 down-regulation. Furthermore, up-regulation of TfR expression in leukemia cells by iron chelator deferoxamine resulted in a further increase in antisense effect (up to 79% Bcl-2 reduction in K562 at the mRNA level) and in caspase-dependent apoptosis (by approximately 3-fold) by Tf-LN. Tf-LN-mediated delivery combined with TfR up-regulation by deferoxamine appears to be a potentially promising strategy for enhancing the delivery efficiency and therapeutic efficacy of antisense oligonucleotides.

A single anti-microRNA antisense oligodeoxyribonucleotide (AMO) targeting multiple microRNAs offers an improved approach for microRNA interference.

Anti-miRNA antisense inhibitors (AMOs) have demonstrated their utility in miRNA research and potential in miRNA therapy. Here we report a modified AMO approach in which multiple antisense units are engineered into a single unit that is able to simultaneously silence multiple-target miRNAs, the multiple-target AMO or MTg-AMO. We validated the technique with two separate MTg-AMOs: anti-miR-21/anti-miR-155/anti-miR-17-5p and anti-miR-1/anti-miR-133. We first verified the ability of the MTg-AMOs to antagonize the repressive actions of their target miRNAs using luciferase reporter activity assays and to specifically knock down the levels of their target miRNAs using real-time RT-PCR methods. We then used the MTg-AMO approach to identify several tumor suppressors-TGFBI, APC and BCL2L11 as the target genes for oncogenic miR-21, miR-155 and miR-17-5p, respectively, and two cardiac ion channel genes HCN2 (encoding a subunit of cardiac pacemaker channel) and CACNA1C (encoding the alpha-subunit of cardiac L-type Ca(2+) channel) for the muscle-specific miR-1 and miR-133. We further demonstrated that the MTg-AMO targeting miR-21, miR-155 and miR-17-5p produced a greater inhibitory effect on cancer cell growth, compared with the regular single-target AMOs. Moreover, while using the regular single-target AMOs excluded HCN2 as a target gene for either miR-1 or miR-133, the MTg-AMO approach is able to reveal HCN2 as the target for both miR-1 and miR-133. Our findings suggest the MTg-AMO as an improved approach for miRNA target finding and for studying function of miRNAs. This approach may find its broad application for exploring biological processes involving multiple miRNAs and multiple genes.