The major issue in the development of nucleic acid based therapeutics is the inefficient delivery of these agents into cells. We prepared cholesterol conjugated spermine and evaluated its usefulness as a delivery modality for antisense oligonucleotides in HeLa-Luc cells. A 2'-O-methyl antisense oligonucleotide sequence, designed to correct splicing at an aberrant intron inserted into a normal luciferase reporter gene, was used for complex formation with cholesterol conjugated spermine. Effective delivery of this antisense agent into nucleus would results in the expression of a luciferasereporter gene product. The cholesterol-spermine formed stable complexes with the antisense oligonucleotide and showed modest delivery activity. Furthermore, this delivery activity was maintained even in the presence of serum proteins, mimicking in vivo conditions. Cholesterol-spermine thus has potential as a delivery system for antisense oligonucleotides into cells.
Blood Proteins ; Cholesterol* ; Genes, Reporter ; Introns ; Luciferases ; Oligonucleotides, Antisense ; Spermine*

Despite advances in neurosurgery, radiation, and chemotherapy, the prognosis of patients with malignant brain tumors still remains grim. Considerable efforts have been made to develop new therapeutic strategies for malignant brain tumors. One of the promising new therapies for brain tumors is an intervention at molecular level, and several molecular approaches have been shown to have in vitro and in vivo activities. These include the use of retroviral vectors, herpes simplex viruses, adenoviral vectors in gene transfer, and antisense vectors and oligonucleotides. Preclinical studies of retroviral vector have already been extended to clinical trials, clearly demonstrating the clinical potential of these molecular therapies. Here, I discuss the current status of molecular therapy for brain tumors together with future directions for its development.
Brain Neoplasms* ; Brain* ; Drug Therapy ; Humans ; Neurosurgery ; Oligonucleotides ; Oligonucleotides, Antisense* ; Prognosis ; Simplexvirus ; Zidovudine

OBJECTIVETo test the efficiency of transfecting (99)Tc(m)-labeled anti-miR208b oligonucleotide into early hypertrophic cardiac myocytes in vitro.

METHODSThe anti-oligonucleotide targeting miR208b (AMO) was synthesized and modified with LNA followed by conjugation with N-hydroxysuccinimidyl S-acetyl-meraptoacetyl triglycine (NHS-MAG3) and radiolabeling with (99)Tc(m). NHS-MAG3-LNA-AMO and labeled AMO were purified with Sep-Pak C18 column chromatography, and the former was examined for UV absorption at the 260 nm using Gene Quant DNA/RNA calculator. The labeling efficiency, radiochemical purity, stability and molecular hybridization activity were analyzed. An angiotensin II-induced cell model of hypertrophic cardiac myocytes was transfected with (99)Tc(m)-NHS-MAG3-LNA-AMO via liposome, and the relative expression of miRNA208b and retention ratio of the labeled AMO in early hypertrophic cells were determined.

RESULTSThe labeling efficiency and radiochemical purity of the labeled AMO after purification exceeded 84% and 86%, respectively. The radio- chemical purities of the labeled AMO incubated in serum and normal saline for 12 h were both higher than 80%, and the labeled AMO showed a capacity to hybridize with the target gene. In the hypertrophic model of cardiac myocytes, the retention ratio of labeled AMO at 6 h was higher than 20%.

CONCLUSIONThe (99)Tc(m)-labeled antisense probe can be efficiently transfected into hypertrophic cardiac myocytes in vitro, which provides an experimental basis for subsequent radionuclide imaging studies.

Humans ; Isotope Labeling ; Liposomes ; MicroRNAs ; genetics ; Myocytes, Cardiac ; Oligonucleotides ; Oligonucleotides, Antisense ; Oligopeptides ; Radiopharmaceuticals ; Silicon Dioxide ; Succinimides ; Transfection

With the rapid development of genetic engineering and metabolic regulation, antisense technology displays its fascination to the world as a mild regulation genetic tool. Compared with other loss-of-function research methods (e.g. gene knockout), antisense technologies have advantages such as low cost, short period, and easy operation. It has been increasingly used in bacterial metabolic regulation as a powerful genetic tool. This review briefly summarized the latest progress and problems in antisense technologies that are recently used in metabolic engineering of bacteria, and compares the advantages and disadvantages of these technologies.
Bacteria ; genetics ; metabolism ; Genes, Bacterial ; Genetic Engineering ; Metabolic Networks and Pathways ; genetics ; Oligonucleotides, Antisense ; genetics ; RNA, Antisense ; genetics ; RNA, Catalytic ; genetics

PURPOSE: Aberrant expression of the c-myb gene is often detected in transformed leukemic cells. Inhibition of c-myb expression by antisense oligos could be an effective way to abort rapid growth of leukemic cells. Developing stable antisense oligos combined with enhanced delivery into cells would be of great use in developing an effective anti-cancer molecular agent. MATERIALS AND METHODS: Selection of target sites was carried out by employing computer simulation of mRNA secondary structures. Multiple antisense oligo sequences were adjoined and AS-oligos were then covalently closed to evade exonuclease activities. C-myb antisense oligos with a novel structure were complexed with cationic liposomes and used to treat HL-60 leukemic cells. RESULTS: We developed covalently closed antisense oligos which harbor four adjoined antisense sequences. The c-myb antisense oligos were found to be exceptionally stable and effective in specifically ablating c-myb mRNA. The antisense oligos were able to inhibit growth of leukemic cell line (HL-60) by about 80%. Antisense effect was more pronounced when the cells were treated twice with the antisense oligos at lower concentrations. CONCLUSION: The novel covalently closed antisense oligo (CMAS-oligos) was found to be effective and exceptionally stable, Growth of HL-60 was significantly inhibited, showing a rational way to develop an effective molecular anti-cancer agent.
Cell Line ; Computer Simulation ; Genes, myb ; Liposomes ; Oligonucleotides, Antisense* ; RNA, Messenger

Ceramide induces cell death in a dose- and time-dependent manner in neuroblastoma SK-N-SH cells. To investigate the mechanism of SK-N-SH cell death by C2-ceramide, morphological features and Hoechst 33258 staining were analyzed. In these morphlogic study the cell death by ceramide showed typical apoptotic features, nuclear condensation, fragmentation, and membrane blebbing. Ceramide-induced apoptosis was accompanied by nuclear accumulation of p53. Inhibition of p53 expression with p53 antisense oligonucleotides inhibited apoptosis evoked by ceramide. Also, ceramide induced mitochondrial event, collapse of mitochondrial membrane potential (delta psi m) and interestingly, inhibition of p53 attenuated collapse of mitochondrial membrane potential, suggests that ceramide induces mitochondrial dysfunction through upregulation of p53 expression. These results suggest that ceramide-induced apoptosis is dependent upon increase in cellular p53 levels which play a critical role in the regulation of apoptotic cell death and p53 modulates mitochondrial function such as mitochondrial membrane potential level.
Apoptosis ; Bisbenzimidazole ; Blister ; Cell Death* ; Membrane Potential, Mitochondrial ; Membranes ; Neuroblastoma ; Neurons* ; Oligonucleotides, Antisense ; Up-Regulation

DNA, Viral ; genetics ; Hep G2 Cells ; Hepatitis B virus ; drug effects ; Humans ; Oligonucleotides, Antisense ; pharmacology

Duchenne muscular dystrophy (DMD) is a lethal muscular disorder caused by mutations in the dystrophin gene for which no mutation targeted therapy has been available so far. However, a new method named exon-skipping mediated by antisense oligonucleotides has considerable potential for DMD therapy. In this review, the principle, basic research and clinical research of exon-skipping are mainly summarized.
Animals ; Exons ; genetics ; Humans ; Muscular Dystrophy, Duchenne ; genetics ; therapy ; Oligonucleotides, Antisense ; genetics

The study was supposed to investigate the inhibitory effect of antisense phosphorothioate oligodeoxynucleotide (ASPSODN) targeting hTERT mRNA on gene of interest in K562 cells and influence of ASPSODN on telomerase activity and apoptosis of K562 cells. Human leukemia cell line K562 was transfected by liposome with ASPSODN and SPSODN (sense phosphorothioate oligodeoxynucleotide) at different concentrations (0.2, 0.6, 1.0 micromol/L). At the same time, blank control, liposome control and SPSODN groups were designed for comparison. The transfected cells were collected and detected at 24 and 48 hours; the expression of target gene hTERT mRNA and telomerase activity were detected by RT-PCR and TRAP-ELISA respectively, and cell apoptosis was assayed by flow cytometry. The results showed that after K562 cells were transfected for 24 hours, the expression of hTERT mRNA had no difference between liposome control (0.80+/-0.24), 0.2 micromol/L ASPSODN (0.69+/-0.12), 0.2 micromol/L SPSODN (0.72+/-0.25) and blank control (0.85+/-0.28), but the expression of hTERT mRNA in 0.6 micromol/L ASPSODN group (0.42+/-0.16) remarkably decreased as compared with liposome control group, 0.6 micromol/L SPSODN (0.69 +/- 0.26) had no obvious effect on the expression of hTERT mRNA, the expression of hTERT mRNA in 1.0 micromol/L ASPSODN and SPSODN groups both decreased; mortality of K562 cells transfected by liposome with 1.0 micromol/L ASPSODN and SPSODN remarkably increased. After 24 hours, telomerase relative activity of K562 cells showed no significant difference between blank control (88.9%) and liposome control (77.7%). The telomerase relative activities of K562 cells treated with 0.2, 0.6, 1.0 micromol/L ASPSODN were 60.6%, 52%, 58.2% respectively. There was significant difference as compared with blank control; 0.6 micromol/L ASPSODN showed significant difference (p=0.037), as compared with liposome control group. The telomerase relative activities in K562 cells treated with 0.2, 0.6, 1.0 micromol/L SPSODN were 76.1%, 72.2%, 65.7% respectively, but the telomerase relative activities of K562 cells in 0.2, 0.6 micromol/L SPSODN groups was not inhibited obviously. When K562 cells were treated for 48 hours, telomerase relative activity of K562 cells in each ASPSODN groups restored. It showed that telomerase relative activities of K562 cells treated with 0.2, 0.6, 1.0 micromol/L ASPSODN were 84.1%, 82.3%, 79.6% respectively, while telomerase relative activities of K562 cells treated with 0.2, 0.6, 1.0 micromol/L SPSODN for 48 hours were 74.8%, 74.5%, 67.9% respectively. Telomerase activity of K562 cells could not be inhibited by 0.2 and 0.6 micromol/L SPSODN. After culturing for 48 hours, the cell apoptosis rates of K562 in 0.6 micromol/L ASPSODN, 0.6 micromol/L SPSODN, liposome control and blank control groups were (4.82+/-0.39)%, (1.83+/-0.34)%, 1.84+/-1.04)%, (1.07+/-0.74)% respectively. There was difference between ASPSODN and SPSODN groups (p<0.05), but the significant difference was found in ASPSODN group as compared with liposome control and blank control (p<0.01). It is concluded that the ASPSODN targeting hTERT can specifically inhibit the expression of hTERT mRNA in K562 cells and significantly suppress the telomerase activity of K562 cells at 0.6 micromol/L, which inhibitory time is short. The ASPSODN at high concentration (1.0 micromol/L) shows definite cytotoxicity. 0.6 micromol/L of ASPSODN significantly induces cell apoptosis, while no such effect was seen in SPSODN group.
Apoptosis ; drug effects ; Humans ; K562 Cells ; Oligonucleotides, Antisense ; pharmacology ; Telomerase ; genetics ; metabolism

Antisense oligonucleotides (ASODN) for therapy is a genetic technology which is based on the base-complementary principle. DNA or RNA sequence synthesized by biotechnology is transferred into the target cells to form mRNA-DNA or mRNA-RNA double strand for inhibiting the expression of target genes. In this way we can control and treat some diseases. The development of antisense oligonucleotides drugs has opened a new area of genetic pharmacology. This paper reviews its classifications, mechanics and its wide application in the treatment of viral infection, tumor and cardiovascular diseases. At the same time we pose the problems that need solving.
Arterial Occlusive Diseases ; therapy ; Humans ; Neoplasms ; therapy ; Oligonucleotides, Antisense ; pharmacology ; therapeutic use ; Virus Diseases ; therapy