Cellular delivery of antisense oligonucleotides.

Antisense oligonucleotides can be successfully employed to inhibit specifically gene expression. However, many oligonucleotide classes are polyanions and cannot passively transit the cell membrane. Thus, the use of naked oligonucleotides for antisense purposes poses some rather stringent challenges, and it is not a trivial task to appropriately interpret the data derived from experiments in which they have been used. Multiple methods have been developed to improve intracellular, and in particular, intranuclear oligonucleotide delivery, and in doing so, to maximize the performance of the antisense technologies that are currently available. This review discusses the use of cationic lipids, protein and peptide delivery agents, and several novel chemical and viral methods that have recently been explored as delivery vehicles, focussing not only on their strengths, but also on their limitations.

Uptake of oligonucleotide-loaded nanoparticles in prostatic cancer cells and their intracellular localization.

The development of antisense biotechnology is dependent, in part, on creating improved methods for delivering oligonucleotides to cells. In this study, we investigated a colloidal system (nanoparticles (NP) of poly (D,L) lactic acid) that affects the intracellular delivery of oligonucleotides. We have examined the intracellular compartmentalization in DU145 cells of fluorescein labeled phosphorothioate oligonucleotides, both in the free state and when loaded into NP. Fluorescent oligonucleotides were incubated for 18 h with DU145 cells and the mean intracellular fluorescence was determined by flow cytometry. After the addition of monensin, an increase in signal intensity was observed, indicating that free oligonucleotides were resident in an acidic intracellular environment, whereas oligonucleotides from the NP did not reside in an acidic compartment. Free and NP loaded with oligonucleotides effluxed from DU145 cells from two intracellular compartments. This preliminary report indicates that colloidal carriers such as NP could prove to be useful in affecting intracellular trafficking of oligonucleotides in DU145 and in other cells.

Highly loaded nanoparticulate carrier using an hydrophobic antisense oligonucleotide complex.

Antisense oligonucleotides, and particularly those with phosphorothioate backbones, have emerged as potential gene specific therapeutic agents and are currently undergoing evaluation in clinical trials for a variety of diseases. In the area of HIV-1 therapeutics, targeting of oligonucleotides to infected cells, such as macrophages, would be highly desirable. The present study was designed to prepare and characterize oligonucleotide-loaded nanoparticles for this purpose. Due to their hydrophilic characteristics, oligonucleotides are difficult to entrap in polymeric particles. Here, the oligonucleotides were first complexed with cetyltrimethylammonium bromide. The oligonucleotide-loaded nanoparticles were prepared by the emulsification-diffusion method and subsequently purified. In comparison with previous studies, a high oligonucleotide-loading was achieved; 2.5, 5 and 10% oligonucleotide loading were assessed. If the initial oligonucleotide content was 4%, this method produced a final oligonucleotide loading of 1.9% with an entrapment efficiency of 47%. The integrity of the oligonucleotide and of the polymer, in the final freeze-dried product, was retained.

Development and antitumor activity of a BCL-2 targeted single-stranded DNA oligonucleotide.

PNT100 is a 24-base, chemically unmodified DNA oligonucleotide sequence that is complementary to a region upstream of the BCL-2 gene. Exposure of tumor cells to PNT100 results in suppression of proliferation and cell death by a process called DNA interference. PNT2258 is PNT100 that is encapsulated in protective amphoteric liposomes developed to efficiently encapsulate the PNT100 oligonucleotide, provide enhanced serum stability, optimized pharmacokinetic properties and antitumor activity of the nanoparticle both in vivo and in vitro. PNT2258 demonstrates broad antitumor activity against BCL-2-driven WSU-DLCL2 lymphoma, highly resistant A375 melanoma, PC-3 prostate, and Daudi-Burkitt's lymphoma xenografts. The sequence specificity of PNT100 was demonstrated against three control sequences (scrambled, mismatched, and reverse complement) all encapsulated in a lipid formulation with identical particle characteristics, and control sequences did not demonstrate antiproliferative activity in vivo or in vitro. PNT2258 is currently undergoing clinical testing to evaluate safety and antitumor activity in patients with recurrent or refractory non-Hodgkin's lymphoma and additional studies are planned.

Phosphorothioate oligonucleotides reduce mitochondrial outer membrane permeability to ADP.

G3139, an antisense Bcl-2 phosphorothioate oligodeoxyribonucleotide, induces apoptosis in melanoma and other cancer cells. This apoptosis happens before and in the absence of the downregulation of Bcl-2 and thus seems to be Bcl-2-independent. Binding of G3139 to mitochondria and its ability to close voltage-dependent anion-selective channel (VDAC) have led to the hypothesis that G3139 acts, in part, by interacting with VDAC channels in the mitochondrial outer membrane (21). In this study, we demonstrate that G3139 is able to reduce the mitochondrial outer membrane permeability to ADP by a factor of 6 or 7 with a K(i) between 0.2 and 0.5 microM. Because VDAC is responsible for this permeability, this result strengthens the aforesaid hypothesis. Other mitochondrial respiration components are not affected by [G3139] up to 1 microM. Higher levels begin to inhibit respiration rates, decrease light scattering and increase uncoupled respiration. These results agree with accumulating evidence that VDAC closure favors cytochrome c release. The speed of this effect (within 10 min) places it early in the apoptotic cascade with cytochrome c release occurring at later times. Other phosphorothioate oligonucleotides are also able to induce VDAC closure, and there is some length dependence. The phosphorothioate linkages are required to induce the reduction of outer membrane permeability. At levels below 1 microM, phosphorothioate oligonucleotides are the first specific tools to restrict mitochondrial outer membrane permeability.

Phosphorothioate oligonucleotides block the VDAC channel.

Proapoptotic phosphorothioate oligonucleotides such as G3139 (an 18-mer) induce Bcl-2-independent apoptosis, perhaps partly via direct interaction with VDAC and reduction of metabolite flow across the mitochondrial outer membrane. Here, we analyzed the interactions at the molecular level. Ten micromolar G3139 induces rapid flickering of the VDAC conductance and, occasionally, a complete conductance drop. These phenomena occur only when VDAC is in the "open" conformation and therefore are consistent with pore blockage rather than VDAC closure. Blockage occurs preferentially from one side of the VDAC channel. It depends linearly on the [G3139] and is voltage-dependent with an effective valence of -3. The kinetics indicate at least a partial entry of G3139 into VDAC, forming an unstable bound state, which is responsible for the rapid flickering (approximately 0.1 ms). Subsequently, a long-lived blocked state is formed. An 8-mer phosphorothioate, polydeoxythymidine, induces partial blockage of VDAC and a change in selectivity from favoring anions to favoring cations. Thus, the oligonucleotide is close to the ion stream. The phosphodiester congener of G3139 is ineffective at the concentrations used, excluding a general polyanion effect. This shows the importance of sulfur atoms. The results are consistent with a binding-induced blockage rather than a permeation block.

Delivery of G3139 using releasable PEG-linkers: impact on pharmacokinetic profile and anti-tumor efficacy.

In order to overcome the problems of enzymatic degradation and short plasma half life, which can limit the delivery of antisense oligonucleotides, and the potential immuno-stimulatory effects of CpG motifs, we utilized a polyethylene glycol (PEG) technology that employed various releasable linkers (rPEG). 5'-20 kDa-PEGylation of an anti-Bcl-2 5'-aminoalkyl-oligonucleotide with the same sequence as G3139 (Compound 1) did not alter its binding to the heparin-binding protein bFGF, nor the release of cytochrome c from isolated mitochondria treated with the conjugates. However, in 518A2 melanoma cells in vitro, PEGylation resulted in greatly diminished cellular uptake. In striking contrast, PEGylation of 1 resulted in dramatically improved pharmacokinetic profiles in vivo, with a prolonged half-life (t1/2), increased plasma concentration, and increased area under the plasma concentration-time curve (AUC). In an in vivo melanoma 518A2 xenograft mouse model, treatment with either 5'-20 kDa-PEG-1 or 1 demonstrated similar tumor growth inhibition. Furthermore, in an in vitro mouse splenocyte culture system, attachment of a PEG moiety to 1 through releasable linkers abolished the immunostimulatory response that was observed for G3139. Our results demonstrate the potential of the in vivo use of PEGylated oligonucleotides, and point out the profound differences between in vitro and in vivo models of oligonucleotide activity.

Inhibition of HIV-1 in cell culture by oligonucleotide-loaded nanoparticles.

PURPOSE: To investigate the potential use of polymeric nanoparticles for the delivery of antisense oligonucleotides in HIV-1-infected cell cultures. METHODS: Phosphorothioate oligonucleotides were encapsulated into poly (D,L-lactic acid) nanoparticles. Two models of infected cells were used to test the ability of nanoparticles to deliver them. HeLa P4-2 CD4+ cells, stably transfected with the beta-galactosidase reporter gene, were first used to evaluate the activity of the oligonucleotides on a single-round infection cycle. The acutely infected lymphoid CEM cells were then used to evaluate the inhibition of the viral production of HIV-1 by the oligonucleotides. RESULTS: The addition to infected CEM cells of nanoparticles containing gag antisense oligonucleotides in the nanomolar range led to strong inhibition of the viral production in a concentration-dependent manner. Similar results were previously observed in HeLa P4-2 CD4+ cells. Nanoparticle-entrapped random-order gag oligonucleotides had similar effects on reverse transcription. However, the reverse transcriptase activity of infected cells treated with nanomolar concentrations of free antisense and random oligonucleotides was not affected. CONCLUSIONS: These results suggest that poly (D,L-lactic acid) nanoparticles may have great potential as an efficient delivery system for oligonucleotides in HIV natural target cells, i.e., lymphocytic cells.

Inhibitory nonsequence-specific effects of cytidine homopolymers on in vivo neointimal formation.

Phosphorothioate oligodeoxynucleotides (PS oligos) manifest both antisense and G-quartet aptameric inhibitory effects on vascular smooth muscle cell (SMC) proliferation. In this study, we examined the effects of three cytidine (S-dC) homopolymers lacking any guanosines of various chain length-S-dC28, S-dC18 and S-dC12-on in vitro SMC proliferation and in vivo neointimal formation. S-dC18 significantly inhibited human vascular SMC proliferation, although it had only half the potency as the same dose of S-dC28. Furthermore, S-dC12 at the same concentrations as S-dC18 did not significantly inhibit vascular SMC proliferation. S-dC28 and S-dC18 inhibited PDGF-induced in vitro SMC migration, whereas D-dC12 had no significant effect on PDGF-induced in vitro SMC migration. We determined the effects of S-dC28, S-dC18, and S-dC12 on neointimal SMC formation in the rat carotid balloon injury model. Rat carotid artery neointimal formation after balloon injury was significantly attenuated by S-dC28 treatment compared with the control group and by S-dC18 treatment compared with the control group. S-dC28 and S-dC18 treatment significantly reduced the intima/media area ratio compared with the values of the control groups. However, S-dC12 did not significantly inhibit neointimal formation. We investigated the time course of the inhibitory effects of S-dC28 on rat carotid artery neointimal formation. S-dC28 significantly inhibited rat carotid artery intimal area and intima/media area ratio at 4 weeks and 8 weeks. Fluoresceinated S-dC28 (FITC-S-dC28) was found to be present throughout the rat carotid arterial wall within 6 hours after balloon injury. Taken together, the potent non-G-quartet, nonsequence-specific inhibitory effects of S-dC compounds on in vitro SMC proliferation and in vivo neointimal formation in the rat carotid balloon injury model are chain length dependent and long lasting.