Cellular uptake of ODNs in HIV-1 human-infected cells: a role for viral particles in DNA delivery?

We have previously described how a 16 nucleotides ODN (termed 93del) is capable of inhibiting the activity of recombinant integrase in a cell-free system as well as HIV-1 replication in human-infected cells with IC(50) in the low nanomolar range. Intracellular HIV-1 replication was inhibited when the ODN was added at the onset of infection. These results raise several questions. Is a naked ODN able to enter the cell? Does the virus play a role in ODN entry? The uptake of several ODNs (93del, 60del(sc), TBA, T30923) was evaluated and then tracked by labeling the ODN with a fluorescent dye and assessing its intracellular localization by confocal microscopy. A significant level of cellular uptake of free ODN was observed in several cell lines: HeLa epithelial cells, Huh7 hepatic cells, and H9 lymphocytes, and was detected for all ODNs tested except for TBA. Striking differences were observed when naked ODNs were added to cell in the presence or absence of the virus. When HIV-1 virions were present a sharp increase in cellular fluorescence was observed. These results strongly suggest a role for HIV-1 virions in the uptake of certain ODNs.     

Highly efficient cellular uptake of c-myb antisense oligonucleotides through specifically designed polymeric nanospheres.

c-myb antisense oligonucleotides (AS ODNs) were reversibly immobilized to a novel polymeric core shell nanosphere and their cellular uptake and inhibitory effect on HL60 leukemia cell proliferation studied. The nanosphere surface was so designed as to directly bind ODNs via ionic interactions and reversibly release them inside the cells. Compared with the cellular uptake of free oligonucleotide, the use of AS ODN (immobilized to the nanospheres) produced a 50-fold increase in the intracellular concentration. Specifically, a single dose of 320 nM of AS ODN immobilized to the nanospheres was capable of inhibiting HL60 cell proliferation with the same degree of efficiency obtained using a 50-fold higher dose of free AS ODN. Flow cytometric experiments with fluoresceinated ODNs showed a temperature-dependent uptake, which was detectable as early as 2 h after the beginning of treatment. The inhibitory effect on cell proliferation was maintained for up to 8 days of culture. Moreover, the level of c-Myb protein decreased by 24% after 2 days and by 60% after 4 days of treatment, thus indicating a continuous and sustained release of non-degraded AS ODN from the nanospheres inside the cells.     

ODN 491, a novel antisense oligodeoxynucleotide that targets thymidylate synthase, exerts cell-specific effects in human tumor cell lines

Thymidylate synthase (TS) is essential for DNA replication and is a target for cancer chemotherapy. However, toxicity to normal cells and tumor cell drug resistance necessitate development of new therapeutic strategies. One such strategy is to use antisense (AS) technology to reduce TS mRNA and protein levels in treated cells. We have developed oligodeoxynucleotides (ODNs) that target different regions of TS mRNA, inhibit human tumor cell proliferation as single agents, and enhance cytotoxicity of clinically useful TS protein-targeting drugs. Here we describe ODN 491, a novel 20mer AS ODN complementary to a previously untargeted portion of the TS mRNA coding region. AS ODN 491 decreased TS mRNA levels to different degrees in a panel of human tumor-derived cell lines, and induced different physiological effects in a tumor cell line-dependent manner. ODN 491 (like AS TS ODN 83, previously shown to be effective) decreased TS protein levels in HeLa cells with a concomitant increase in sensitivity to TS-targeting chemotherapeutics. However (and contrary to HeLa cell response to an AS ODN 83), it did not, as a single agent, inhibit HeLa cell proliferation. In MCF-7 cells, ODN 491 treatment was less effective at reducing TS mRNA and did not reduce TS protein, nor did it enhance sensitivity to TS-targeting or other chemotherapeutics. Moreover, specifically in MCF-7 cells but not HeLa cells, ODN 491 as a single agent induced apoptosis. These data indicate that AS TS ODN 491 is an effective AS reagent targeting a novel TS mRNA region. However, treatment of tumor cell lines with AS TS ODNs targeting different TS mRNA regions results in a pattern of physiological effects that varies in a tumor cell line-specific fashion. In addition, the capacity of different AS TS ODNs to induce physiological effects does not correlate well with their capacity to reduce TS mRNA and/or protein and, further, depends on the region of TS mRNA selected for targeting. Recognition of tumor cell-specific and mRNA region-specific variability in response to AS TS ODNs will be important in designing AS TS ODNs for potential clinical use.     

Progress in the delivery of therapeutic oligonucleotides: organ/cellular distribution and targeted delivery of oligonucleotides in vivo

Oligonucleotide (ODN) therapy is a powerful tool for modulation of gene expression in vivo. With advances in ODN chemistry and progress in formulation development, ODNs are becoming widely acceptable drugs. This review summarizes the current status and future trend of the in vivo application of ODN therapeutics, especially antisense ODNs. Here, we review the current understanding of the tissue/organ distribution and cellular uptake of ODN drugs administered parenterally or nonparenterally to intact animals. The problems and advantages inherent in the use of different delivery methods for the treatment of particular diseases are discussed in detail. Emphasis is placed on the most widely studied ODN analogs, the phosphorothioates (PS). Lessons learned from antisense PS studies have broad implications for ODN therapeutics in general.     

Crystallization of human c-H-ras oncogene products

There is compelling evidence that cancer develops as a consequence of genetic changes (probably multiple) in some members of a selected set of cellular genes. DNA isolated from a variety of tumors, but not normal tissues, possesses the ability to malignantly transform non-tumorigenic cells. Many oncogenes responsible for such transformation have been isolated from transformed cell lines and animal and human tumors induced spontaneously, by virus, by chemical, or by radiation. The most commonly found transforming genes isolated from human tumor cells by DNA transfection assay are the ras gene family (c-H-ras, c-K-ras and N-ras). We report crystallization of several human c-H-ras oncogene proteins.     

Microbubble-enhanced ultrasound to deliver an antisense oligodeoxynucleotide targeting the human androgen receptor into prostate tumours

We have shown recently that downregulation of the androgen receptor (AR), one of the key players in prostate tumor cells, with short antisense oligodeoxynucleotides (ODNs) results in inhibition of prostate tumor growth. Particularly with regard to an application of these antisense drugs in vivo, we now investigated the usefulness of microbubble-enhanced ultrasound to deliver these ODNs into prostate cancer cells.

Our short antisense AR ODNs were loaded onto the lipid surface of cationic gas-filled microbubbles by ion charge binding, and delivered into the cells by bursting the loaded microbubbles with ultrasound. In vitro experiments were initially performed to show that this kind of delivery system works in principle. In fact, transfection of prostate tumor cells with antisense AR ODNs using microbubble-enhanced ultrasound resulted in 49% transfected cells, associated with a decrease in AR expression compared to untreated controls. In vivo, uptake of a digoxigenin-labelled ODN was found in prostate tumour xenografts in nude mice following intratumoral or intravenous injection of loaded microbubbles and subsequent exposure of the tumour to ultrasound, respectively. Our results show that ultrasound seems to be the driving force of this delivery system. Uptake of the ODN was also observed in tumors after treatment with ultrasound alone, with only minor differences compared to the combined use of microbubbles and ultrasound.     

Cellular ras activity is required for passage through multiple points of the G0/G1 phase in BALB/c 3T3 cells.

Microinjection experiments demonstrated a requirement for cellular ras activity late in G1. In this study, we used two separate methods to identify an additional requirement for cellular ras activity early in the G0/G1 phase of the cell cycle. Quiescent BALB/c cells were injected with anti-ras antibody prior to stimulation with serum. The cells would therefore be inhibited in progression through the cell cycle at the earliest point requiring ras function. Alternatively, cells were inhibited in late G1 as in previous studies by injecting anti-ras several hours after serum addition to quiescent cells. The injected cultures were then treated with chemical cell cycle inhibitors known to function in mid-G1. Cells injected with anti-ras prior to serum stimulation were retained at a point of ras requirement prior to the execution point of the chemical inhibitor, while cells injected 3 to 5 h after serum stimulation were retained at a point of ras requirement downstream of the execution point of the chemical inhibitor. To confirm these results, quiescent BALB/c cells were injected with anti-ras antibody prior to or several hours following serum addition. In this case, however, second injections of oncogenic ras or adenoviral E1A protein were performed to overcome the inhibitory effects of the anti-ras antibody. Cells injected prior to serum addition were clearly inhibited at an early point of Ras requirement since they required 5 or 6 h longer to enter S phase than cells injected with anti-ras antibody after serum addition.     

In vivo pro-apoptotic and antitumor efficacy of a c-Raf antisense phosphorothioate oligonucleotide: relationship to tumor size

Previously, we have shown that a phosphorothioate antisense oligonucleotide (ODN) targeted against c-raf RNA (ISIS5132; cRaf-AS) induces apoptosis in human tumor cells. We now show that the same ODN also efficiently triggers apoptosis in human tumor xenografts in nu/nu mice. Although cRaf-AS showed a clearly inhibitory effect on the growth of established tumors (approximately 150 mm3) compared to a mismatched control ODN (MM), tumor progression was not prevented. This correlated with a partial refractoriness of the tumor to cRaf-AS-induced cell killing, which seemed to be due to an inhomogeneous and inefficient penetration of the ODN into the tumor tissue rather than cellular resistance. In agreement with this conclusion, we found that growth of small tumors (<50 mm3) was completely inhibited concomitantly with an accumulation of the ODN throughout the tumor. These data show that the cRaf-AS is a highly efficacious antitumor agent, provided accessibility into the tumor tissue is warranted, and suggest that PS-AS-ODN treatment may be particularly useful in an adjuvant setting.     

Sweet delivery - sugar translocators as ports of entry for antisense oligodeoxynucleotides in plant cells

Antisense oligodeoxynucleotides (ODNs) are short (12-25 nt long) stretches of single-stranded DNA that may be delivered to a cell, where they hybridize to the cognate mRNA in a sequence-specific manner, thereby inhibiting gene expression. Here we used confocal microscopy to monitor the uptake and trafficking of ODNs in barley tissues. We conclude that uptake of ODNs across the plant plasma membrane is mediated by active transport of mono- or disaccharides through sugar translocators. We demonstrate that sugar transport can deliver ODNs to barley seeds, and that this strategy may be employed to suppress gene activity in endosperm cells by antisense ODN inhibition. We further found that sucrose compared favorably with oligofectamine as a vehicle for ODN delivery to human cells in a low-serum environment.     

Electroporation enhances c-myc antisense oligodeoxynucleotide efficacy.

Obtaining high transfection efficiencies and achieving appropriate intracellular concentrations and localization are two of the most important barriers to the implementation of gene targeted therapy. The efficiency of endogenous uptake of oligodeoxynucleotides (ODNs) varies from cell type to cell type and may be a limiting factor of antisense efficacy. The use of electroporation to obtain high intracellular concentrations of a synthetic ODN in essentially 100% of viable cells is described. It is also shown that the transfected ODNs initially localize to the nucleus and remain there for at least 48 hours. The cellular trafficking of electroporated ODNs is shown to be an energy dependent process. Targeting of the c-myc proto-oncogene of U937 cells by electroporation of phosphorothioate-modified ODNs results in rapid and specific suppression of this gene at ODN concentrations much lower than would otherwise be required. This technique appears to be applicable to a variety of cell types and may represent a powerful new investigate tool as well as a promising approach to the ex vivo treatment of hematologic disorders.     

The adenovirus E1A protein overrides the requirement for cellular ras in initiating DNA synthesis.

The adenovirus E1A protein can induce cellular DNA synthesis in growth-arrested cells by interacting with the cellular protein p300 or pRb. In addition, serum- and growth factor-dependent cells require ras activity to initiate DNA synthesis and recently we have shown that Balb/c 3T3 cells can be blocked in either early or late G1 following microinjection of an anti-ras antibody. In this study, the E1A 243 amino acid protein is shown through microinjection not only to shorten the G0 to S phase interval but, what is more important, to override the inhibitory effects exerted by the anti-ras antibody in either early or late G1. Specifically, whether E1A is co-injected with anti-ras into quiescent cells or injected 18 h following a separate injection of anti-ras after serum stimulation, it efficiently induces cellular DNA synthesis in cells that would otherwise be blocked in G0/G1. Moreover, injection of a mutant form of E1A that can no longer associate with p300 is just as efficient as wild-type E1A in stimulating DNA synthesis in cells whose ras activity has been neutralized by anti-ras. The results presented here show that E1A is capable of overriding the requirement of cellular ras activity in promoting the entry of cells into S phase. Moreover, the results suggest the possibility that pRb and/or pRb-related proteins may function in a ras-dependent pathway that enables E1A to achieve this activity.     

Poly(propylacrylic acid) enhances cationic lipid-mediated delivery of antisense oligonucleotides

The use of antisense oligodeoxynucleotides (ODNs) to inhibit the expression of specific mRNA targets represents a powerful technology for control of gene expression. Cationic lipids and polymers are frequently used to improve the delivery of ODNs to cells, but the resulting complexes often aggregate, bind to serum components, and are trafficked poorly within cells. We show that the addition of a synthetic, pH-sensitive, membrane-disrupting polyanion, poly(propylacrylic acid) (PPAA), improves the in vitro efficiency of the cationic lipid, DOTAP, with regard to oligonucleotide delivery and antisense activity. In characterization studies, ODN complexation with DOTAP/ODN was maintained even when substantial amounts of PPAA were added. The formulation also exhibited partial protection of phosphodiester oligonucleotides against enzymatic digestion. In Chinese hamster ovary (CHO) cells, incorporation of PPAA in DOTAP/ODN complexes improved 2- to 3-fold the cellular uptake of fluorescently tagged oligonucleotides. DOTAP/ODN complexes containing PPAA also maintained high levels of uptake into cells upon exposure to serum. Addition of PPAA to DOTAP/ODN complexes enhanced the antisense activity (using GFP as the target) over a range of PPAA concentrations in both serum-free, and to a lesser extent, serum-containing media. Thus, PPAA is a useful adjunct that improves the lipid-mediated delivery of oligonucleotides.     

Tumor-targeting,systemically delivered antisense HER-2 chemosensitizes human breast cancer xenografts irrespective of HER-2 levels

BACKGROUND: The failure to respond to chemotherapy is a major obstacle in the successful treatment of breast cancer. We have previously shown that anti-HER-2 antisense oligonucleotide (AS HER-2 ODN) treatment was able to sensitize breast cancer cells to various chemotherapeutic agents in vitro irrespective of their HER-2 status, indicating that the use of AS HER-2 ODN therapy for breast cancer is not limited to tumors overexpressing the protein. One of the main drawbacks to the use of antisense therapy in the clinical setting is the lack of an efficient, tumor-targeting, systemic delivery method. We have developed a tumor-specific, ligand-targeting, cationic liposome delivery system designed for systemic gene therapy of cancer. In this study we employ this ligand-liposome strategy to enhance the delivery of the AS Her-2 ODN to breast cancer cells, including those that do not overexpress HER-2, in vitro and in vivo. MATERIALS AND METHODS: A cationic liposome complex that includes folate as the targeting ligand was designed and optimized for more efficient delivery of AS HER-2 ODN to breast tumors cells in vitro, and more significantly, for systemic delivery with tumor-specific targeting in vivo. Human breast cancer cell line MDA-MB-435, which does not overexpress HER-2, was used to compare the degree of chemosensitization to the taxanes of AS HER-2 ODN delivered via the optimized folate-liposome versuscommercial Lipofectin. MDA-MB-435 xenograft tumors were also used to evaluate the anti-tumor effect of the combination of systemically delivered folate-liposome-AS HER-2 ODN and docetaxel (Taxotere). RESULTS: The optimized folate-liposome-AS HER-2 ODN complex significantly increases the response of breast tumor cell lines to conventional chemotherapeutic agents in vitro as compared to AS HER-2 delivered via an unliganded commercially available reagent, Lipofectin. In vivo, the folate-liposome-AS HER-2 ODN complex has prolonged stability in blood and increased uptake in tumors. More significantly, the combination of intravenously administered ligand-liposome-AS HER-2 ODN and docetaxel resulted in a marked inhibition of xenograft growth in an aggressive breast cancer model that does not overexpress HER-2, even after treatment ended. CONCLUSIONS: Although there are other reports of liposomal delivery of AS ODNs, this is the first report of in vivo efficacy against human cancer cells using a tumor-targeting liposome delivery system for systemic AS therapy. Moreover, the increased stability in circulation and anti-tumor efficacy observed were obtained without the need for continuous intravenous infusion. HER-2 is an integral component within a network of cell growth pathways that can affect many different types of tumors where HER-2 may be a contributing factor, such as ovarian, esophageal, and GI malignancies including colon and pancreatic cancers. Therefore, the effectiveness of this therapy with xenograft tumors that do not overexpress HER-2 has the potential to expand the clinical usefulness of this efficacious form of therapy.     

G-rich oligonucleotides alter cell cycle progression and induce apoptosis specifically in OE19 esophageal tumor cells

Short synthetic oligonucleotides (ODNs) can be used to block cellular processes involved in cell growth and proliferation. Often acting as aptamers, these molecules interact with critical proteins that regulate the induction of apoptosis or necrosis. We have used a specialized class of ODNs that contain a monomeric sequence of guanosine to induce apoptosis specifically in the malignant esophageal cell line, OE19, in cell culture, and in a NODscid mouse model. OE19 cells were grown in culture and treated with a stable G-rich oligonucleotide (GRO). Cells were processed and apoptosis was measured by FACS analyses, caspase activity, and Hoescht staining. Circular dichroism (CD) was used to define the structure and stability of various GROs. The GRO works by first inducing retardation in the progression of the cell cycle and then by creating a sub-G1 population of apoptotic cells. The reaction is dose dependent, and appears to rely on the capacity of the G-rich ODN to adopt a G-quartet conformation. Apoptosis was measured by determining caspase 3/7 levels and by staining for nuclear fragmentation using the Hoechst dye. Importantly, nonmalignant esophageal cells or normal human lung fibroblasts are not impeded in their cell cycle progression when incubated with the G-rich ODNs. These results suggest that a selective killing of esophageal tumor cells is directed by G-rich ODNs. Selective killing was demonstrated in the unique activity of the GRO compared to other ODNs of different sequences as well as the response of oncogenic cells compared to nononcogenic cells.     

Oligonucleotide structure influences the interactions between cationic polymers and oligonucleotides

We examined the effect of oligodeoxynucleotide (ODN) structure on the interactions between cationic polymers and ODNs. Unstructured and hairpin structured ODNs were used to form complexes with the model cationic polymer, poly-L-lysine (pLL), and the characteristics of these polymer-ODN interactions were subsequently examined. We found that hairpin structured ODNs formed complexes with pLL at slightly lower pLL:ODN charge ratios as compared to unstructured ODNs and that, at high charge ratios, greater fractions of the hairpin ODNs were complexed, as measured by dye exclusion. The dissociation of pLL-ODN interactions was tested further by challenge with heparin, which induced complex disruption. Both the kinetics and heparin dose response of ODN release were determined. The absolute amount and the kinetic rate of ODN release from the complexes of pLL and unstructured ODN were greater, as compared to hairpin ODNs. Our results therefore highlight the role of ODN structure on the association-dissociation behavior of polymer-ODN complexes. These findings have implications for the selection of ODN sequences and design of polymeric carriers used for cellular delivery of ODNs.     

H-ras activation in benign and self-regressing skin tumors (keratoacanthomas) in both humans and an animal model system.

The involvement of the ras oncogenes in tumorigenesis was investigated in keratoacanthomas, which are benign and self-regressing skin tumors, both in humans and in a corresponding animal model system. Keratoacanthomas were induced on rabbit ears by repeated applications of 7,12-dimethylbenz(a)anthracene. About 60% of the tumor DNAs produced transformed foci after transfection into NIH 3T3 cells, and in all of them the transforming gene was identified as H-ras by Southern and Northern (RNA) hybridization. Immunoprecipitation experiments suggested that the transforming rabbit H-ras protein carried a mutation in codon 61. In addition, an activated H-ras gene was detected in a human keratoacanthoma by using a nude mouse tumorigenesis assay after transfection of tumor DNA into NIH 3T3 cells. This is the first report of ras activation in a benign human tumor. The transforming human H-ras gene showed a point mutation in codon 61 that would result in leucine instead of the glutamine present in the normal gene product. The finding of ras activation in tumors that are not only benign but also self-regressing indicates that activated ras genes are not sufficient to maintain a neoplastic phenotype, although they likely play a role in early stages of tumorigenesis.     

Psoralen-modified clamp-forming antisense oligonucleotides reduce cellular c-Myc protein expression and B16-F0 proliferation

The c-myc protooncogene plays an important role in the abnormal growth pattern of melanoma cells. In an attempt to inhibit c-Myc expression and the growth of an established murine melanoma cell line, we targeted homopurine sequences within the mouse myc mRNA with modified antisense oligonucleotides (AS ODNs). Psoralen was conjugated to the 5′-end of these clamp-forming oligonucleotides (clamp ODNs). Gel mobility shift analysis demonstrated a sequence-specific interaction between the active clamp ODNs (Myc-E2C and Myc-E3C) and the 1.4 kb c-myc mRNA, but no interaction with the control clamp ODN (SCR**). This association was further confirmed by thermal denaturation studies. In vitro translation assays demonstrated that both Myc-E2C and Myc-E3C at 5 µM inhibited c-Myc expression >99% after UV activation at 366 nm. Immunostaining of B16-F0 cells with a c-Myc monoclonal antibody revealed a significant reduction in c-Myc after clamp ODN treatment compared with the untreated or SCR** control-treated cells. This result was corroborated by western blot analysis. Utilizing the MTT assay to determine the effects of ODN-mediated c-Myc reduction on B16-F0 growth, we observed 60 and 64% reductions in growth after treatment with 5 µM Myc-E3C and Myc-E2C, respectively. We attribute the enhanced effectiveness of the clamp ODNs to psoralen activation. Our preliminary data suggest that inhibiting c-Myc overexpression results in a significant reduction in abnormal proliferation of B16-F0 melanoma cells and that the increased efficiency of clamp ODNs may provide an important advantage for their use in antisense therapies.