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1.
Methods ; 126: 201-208, 2017 08 15.
Article in English | MEDLINE | ID: mdl-28600226

ABSTRACT

The control of RNA processing plays an important role in the nature and quantity of protein generated from mammalian genes. Consequently, efforts to manipulate RNA processing have the capacity to significantly impact gene function. Although multiple strategies have been developed to alter splice site selection using oligonucleotide occlusion of splice sites or splicing regulatory elements, systemic delivery of such agents remains problematic. Outlined in this chapter is a protocol to screen for small molecule inhibitors of HIV-1 Gag expression that have been subsequently determined to modulate viral RNA processing. Identification and characterization of such RNA processing modulators offers the potential for the development of therapeutic lead compounds or probes for investigating the mechanism underlying the regulation of select RNA processing events.


Subject(s)
Gene Expression Regulation, Viral , Genes, gag/physiology , Genetic Testing/methods , HIV-1/metabolism , Animals , Cantharidin/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Genes, gag/drug effects , HIV-1/drug effects , HIV-1/genetics , HeLa Cells , Humans , Pyrans/pharmacology , Spiro Compounds/pharmacology
2.
Drug Deliv ; 23(7): 2596-2607, 2016 Sep.
Article in English | MEDLINE | ID: mdl-26024387

ABSTRACT

In recent years, mRNA-based vaccines have emerged to be a great alternative to DNA-based vaccines due to the safety of not inserting into host genome. However, mRNA molecules are single-stranded nucleic acids that are vulnerable under RNase existing in human skin and tissues. In this study, a self-assembled cationic nanomicelles based on polyethyleneimine-stearic acid (PSA) copolymer were developed to delivery HIV-1 gag encoding mRNA to dendritic cells and BALB/c mice. We evaluated the transfection efficiency and cell uptake efficiency of naked EGFP mRNA, PSA, PEI-2k and PEI-25k nanoparticles format on DC2.4 cell lines. Immune responses after sub-cutaneous administration of gag mRNA to BALB/c mice were notably induced by PSA as compared with naked gag mRNA. We found the PSA/mRNA nanomicelles were potent systems that can effectively deliver mRNA and induce antigen-specific immune response, stimulating various new vaccine strategies using mRNA.


Subject(s)
Cations/chemistry , Dendritic Cells/chemistry , Genes, gag/drug effects , HIV-1/drug effects , RNA, Messenger/immunology , Ribonucleases/immunology , Vaccines/immunology , Animals , Genes, gag/genetics , Genes, gag/immunology , HIV-1/chemistry , HIV-1/immunology , Humans , Mice , Mice, Inbred BALB C , Micelles , RNA, Messenger/chemistry , RNA, Messenger/genetics , Ribonucleases/chemistry , Ribonucleases/metabolism , Stearic Acids/chemistry , Transfection , Vaccines/chemistry , Vaccines/metabolism
3.
Transpl Int ; 27(1): 96-105, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24138389

ABSTRACT

To effectively suppress porcine endogenous retroviruses (PERV)s, RNAi technique was utilized. RNAi is the up-to-date skill for gene knockdown which simultaneously multitargets both gag and pol genes critical for replication of PERVs. Previously, two of the most effective siRNAs (gag2, pol2) were found to reduce the expression of PERVs. Concurrent treatment of these two siRNAs (gag2+pol2) showed knockdown efficiency of up to 88% compared to negative control. However, despite the high initial knockdown efficiency 48 h after transfection caused by siRNA, it may only be a transient effect of suppressing PERVs. The multitargeting vector was designed, containing both gag and pol genes and making use of POL II miR Expression Vector, which allowed for persistent and multiple targeting. This is the latest shRNA system technique expressing and targeting like miRNA. Through antibiotics resistance characteristics utilizing this vector, miRNA-transfected PK15 cells (gag2-pol2) were selected during 10 days. An 88.1% reduction in the level of mRNA expression was found. In addition, we performed RT-activity analysis and fluorescence in situ hybridization assay, and it demonstrated the highest knockdown efficiency in multitargeting (gag2+pol2) miRNA group. Therefore, according to the results above, gene knockdown system (siRNA and shRNA) through multitargeting strategy could effectively inhibit PERVs.


Subject(s)
Endogenous Retroviruses/physiology , RNA Interference , Animals , Cell Line , Coculture Techniques , Gene Knockdown Techniques , Genes, gag/drug effects , Genes, pol/drug effects , HEK293 Cells , Humans , Swine
4.
Biochem Biophys Res Commun ; 313(1): 55-61, 2004 Jan 02.
Article in English | MEDLINE | ID: mdl-14672697

ABSTRACT

Zintevir is an oligonucleotide analogue, which has the phosphorothioate modification at both termini, that forms a K(+)-induced quadruplex structure and shows potent anti-human immunodeficiency virus (HIV)-1 activity. We synthesized the non-modified analogue (D-17mer) of Zintevir and its enantiomer (L-17mer), and compared their anti-HIV-1 activity and molecular mechanism of action. Although L-17mer forms the exact mirror image quadruplex structure of D-17mer, which has a very similar structure with Zintevir, L-17mer showed comparable anti-HIV-1 activity with Zintevir. The results obtained by the time-of-addition experiments and the immunofluorescence binding assay strongly suggest that the primary molecular target of L-17mer is the viral gp120 envelope protein as well as Zintevir, regardless of their reciprocal chirality.


Subject(s)
Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Oligonucleotides/chemistry , Oligonucleotides/pharmacology , Antibodies, Monoclonal/metabolism , Base Sequence , CD4 Antigens/immunology , Cell Line, Transformed , Circular Dichroism , Drug Stability , Flow Cytometry , Genes, gag/drug effects , Genes, gag/genetics , HIV Envelope Protein gp120/immunology , Humans , Nucleic Acid Conformation , Oligonucleotides/chemical synthesis , Oligonucleotides/metabolism , Phosphodiesterase I/metabolism , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Stereoisomerism , Temperature , Time Factors
5.
AIDS Res Hum Retroviruses ; 13(10): 857-63, 1997 Jul 01.
Article in English | MEDLINE | ID: mdl-9197379

ABSTRACT

Thalidomide is currently being evaluated for efficacy in alleviating some manifestations of HIV-1 infection. To determine whether thalidomide has any direct effects on HIV-1 infection, we investigated the effect of thalidomide and also of three structural analogs of thalidomide on HIV-1 replication in vitro in human monocyte-derived macrophages. The thalidomide analogs were previously shown to inhibit TNF-alpha production in vitro at much lower concentrations than thalidomide. In HIV-1-infected macrophages treated with thalidomide or thalidomide analogs, viral replication was reduced by 60 to 80% as determined by measuring viral RT activity in the culture supernatants. In all experiments the analogs inhibited HIV-1 replication more efficiently than did thalidomide. The drugs also reduced HIV-1 gag mRNA expression. Furthermore, the drugs caused a decrease in NF-kappaB-binding activity in nuclear extracts of HIV-1-infected macrophages. The role of NF-kappaB in the drug-induced inhibition of HIV-1 replication was confirmed using an NF-kappaB-defective mutant virus to infect macrophages.


Subject(s)
Anti-HIV Agents/pharmacology , HIV-1/drug effects , HIV-1/physiology , Thalidomide/analogs & derivatives , Thalidomide/pharmacology , Virus Replication/drug effects , Drug Evaluation, Preclinical , Genes, gag/drug effects , HIV Reverse Transcriptase/metabolism , Humans , In Vitro Techniques , Macrophages/drug effects , Macrophages/metabolism , Macrophages/virology , Mutation , NF-kappa B/genetics , NF-kappa B/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism
6.
Antisense Nucleic Acid Drug Dev ; 7(1): 13-22, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9055034

ABSTRACT

A 25-mer phosphorothioate oligodeoxynucleotide (GEM 91) complementary to the gag gene mRNA of HIV-1 virus was administered intravenously (i.v.) at a dose of 10 mg/kg/day for 8 weeks or 25 mg/kg single dose subcutaneously (SC) to adult Rhesus monkeys. No radioactive markers were used. A capillary gel electrophoresis (CGE) method with UV detection was used to determine the concentration of GEM 91 in plasma and the metabolite profile. The metabolite profile was virtually the same following a single dose of either 10 mg/kg i.v. or 25 mg/kg SC. A different metabolite profile was observed after 4 or 8 weeks of multiple i.v. doses of 10 mg/kg/day. The extract was subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) for positive identification. Mass spectrometry confirmed the major metabolic pathway in vivo to be via 3'-end exonuclease activity. The extract was then subjected to a hybridization-assisted ligation reaction in which only 5'-end intact metabolites were labeled. Analysis by CGE with laser-induced fluorescence (LIF) detection allowed each of these metabolites to be quantified with a limit of detection of 1 ppb (ng/ml). MALDI-TOFMS identified components digested from both ends of the DNA. This study demonstrates that the combination of quantitative CGE-LIF and MALDI-TOFMS yields a powerful and unique approach to study the metabolism of phosphorothioate oligonucleotides.


Subject(s)
Oligodeoxyribonucleotides, Antisense , Oligonucleotides, Antisense/administration & dosage , Oligonucleotides, Antisense/metabolism , Thionucleotides/administration & dosage , Thionucleotides/metabolism , Animals , Drug Administration Schedule , Genes, gag/drug effects , Genes, gag/genetics , HIV-1/genetics , HIV-1/metabolism , Humans , Injections, Intravenous , Macaca mulatta , Oligonucleotides, Antisense/blood , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Thionucleotides/blood
7.
Leukemia ; 9 Suppl 1: S86-8, 1995 Oct.
Article in English | MEDLINE | ID: mdl-7475322

ABSTRACT

We have employed a cos-like monkey kidney cell line (B4.14) transfected with plasmids pCMVgag-pol-rre-r (containing the HIV gag and pol genes) and pCMVrev (containing the HIV rev gene), as a model to investigate whether antisense constructs could interfere with specific HIV gene and protein expression. We utilized an antisense construct (GP12A) directed against a non-regulatory region of the HIV genome, to transfect cells that expressed the above-mentioned HIV genes. Our results show that GP12A was able to attenuate levels of relevant HIV mRNA and gag proteins in the absence of cytotoxic effects.


Subject(s)
Antiviral Agents/pharmacology , Gene Expression/drug effects , Gene Products, gag/biosynthesis , Genes, gag/drug effects , HIV-1/genetics , Oligonucleotides, Antisense/pharmacology , Animals , Base Sequence , Cell Line , Chlorocebus aethiops , Genes, pol , Genome, Viral , HIV-1/drug effects , Humans , Kidney , Molecular Sequence Data , Regulatory Sequences, Nucleic Acid , Thionucleotides , Transcription, Genetic/drug effects
8.
Antiviral Res ; 17(1): 53-62, 1992 Jan.
Article in English | MEDLINE | ID: mdl-1736810

ABSTRACT

Sequences from the gag, pol and rev regions of the RF strain of HIV-1 (HIV-1RF) were chosen as targets for antisense phosphorothioate oligodeoxynucleotides (S-oligos). These sequences were the p18/p24 junction in gag, the active site of HIV protease in pol; a sequence from the first exon of the rev gene and S-oligodeoxycytidylic acid controls. Compounds were tested against HIV-1 in both acutely and chronically infected cells. The results show that these phosphorothioate analogues tested in acutely infected cells were active in the 0.1-2 microM range, were dependent on chain length but had no sequence specificity. To study the mechanism of action, the time of addition of S-oligos to acutely infected cells was delayed for up to 48 h post-infection. It was found that antiviral activity was lost when compounds were added to the cultures later than 10 h post-infection. With chronically infected cells only the antisense rev sequence showed activity at 30 microM and neither of the gag or pol antisense sequences has a significant effect on HIV replication at 50 microM. These results are consistent with previous in vitro studies which demonstrate that antisense S-oligodeoxynucleotides have several modes of action.


Subject(s)
Antiviral Agents/pharmacology , Genes, gag/drug effects , Genes, pol/drug effects , Genes, rev/drug effects , HIV-1/drug effects , Oligonucleotides, Antisense/pharmacology , Base Sequence/drug effects , Cell Line , HIV-1/physiology , Humans , Lymphocytes , Molecular Sequence Data , Thionucleotides/pharmacology
9.
AIDS Res Hum Retroviruses ; 7(4): 403-8, 1991 Apr.
Article in English | MEDLINE | ID: mdl-1906290

ABSTRACT

The virucidal efficacy of various commercially available contact lens care cleaning regimens on human immunodeficiency virus (HIV-1) contaminated contact lenses using either cursory or meticulous cleaning with a rubber policeman was evaluated. Levels of infectious HIV-1 remaining on individual contact lenses were determined by cultivating the lenses with target HUT-78 cells and subsequently analyzing the cultures for the production of HIV-1 p24 by antigen capture and for HIV-1 gag gene DNA content by the polymerase chain reaction. The data indicate that most of the lens care regimens tested, when coupled with meticulous rubbing, were capable of safely decontaminating the contact lenses, that is, they reduce the amount of infectious HIV-1 on the lenses by greater than a 10 log concentration (10(-10], relative to standard controls. Most tested lens care regimens, if properly followed, would virtually eliminate any chance of the lens serving as a vector for HIV.


Subject(s)
Contact Lenses, Hydrophilic , Disinfectants/pharmacology , HIV-1/drug effects , Ophthalmic Solutions/pharmacology , DNA, Viral/analysis , Enzyme-Linked Immunosorbent Assay , Gene Products, gag/analysis , Genes, gag/drug effects , HIV Core Protein p24 , HIV-1/genetics , HIV-1/growth & development , Polymerase Chain Reaction , Viral Core Proteins/analysis , Virus Cultivation
11.
Science ; 247(4947): 1222-5, 1990 Mar 09.
Article in English | MEDLINE | ID: mdl-2107573

ABSTRACT

Certain RNA molecules, called ribozymes, possess enzymatic, self-cleaving activity. The cleavage reaction is catalytic and no energy source is required. Ribozymes of the "hammerhead" motif were identified in plant RNA pathogens. These ribozymes possess unique secondary (and possibly tertiary) structures critical for their cleavage ability. The present study shows precise cleavage of human immunodeficiency virus type 1 (HIV-1) sequences in a cell-free system by hammerhead ribozymes. In addition to the cell-free studies, human cells stably expressing a hammerhead ribozyme targeted to HIV-1 gag transcripts have been constructed. When these cells were challenged with HIV-1, a substantial reduction in the level of HIV-1 gag RNA relative to that in nonribozyme-expressing cells, was observed. The reduction in gag RNA was reflected in a reduction in antigen p24 levels. These results suggest the feasibility of developing ribozymes as therapeutic agents against human pathogens such as HIV-1.


Subject(s)
Acquired Immunodeficiency Syndrome/drug therapy , Genes, gag/drug effects , HIV-1/drug effects , RNA, Ribosomal/pharmacology , RNA, Viral/drug effects , Base Sequence , Catalysis , Cloning, Molecular , Gene Expression , Gene Products, gag/metabolism , HIV Core Protein p24 , HIV-1/genetics , HeLa Cells , Humans , Molecular Sequence Data , Nucleic Acid Hybridization , Polymerase Chain Reaction , RNA, Catalytic , RNA, Ribosomal/therapeutic use , Transfection , Viral Core Proteins/metabolism
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