Biomedical subjects

Lun-Quan Sun

Publications and source records attributed to Lun-Quan Sun.

11 recordsLinked to original sources

PubMed2005

Long-term survival and concomitant gene expression of ribozyme-transduced CD4+ T-lymphocytes in HIV-infected patients.

BACKGROUND: An anti-HIV-1 tat ribozyme, termed Rz2, has been shown to inhibit HIV-1 infection/replication and to decrease HIV-1-induced pathogenicity in T-lymphocyte cell lines and normal peripheral blood T-lymphocytes. We report here the results of a phase I gene transfer clinical trial using Rz2. METHODS: Apheresis was used to obtain a peripheral blood cell population from each of four HIV-negative donors. After enrichment for CD4+ T-lymphocytes, ex vivo expansion and genetic manipulation (approximately equal aliquots of the cells were transduced with the ribozyme-containing (RRz2) and the control (LNL6) retroviral vector), these cells were infused into the corresponding HIV-1-positive twin recipient. Marking was assessed over an initial 24-week period and in total over an approximate 4-year period. RESULTS: The gene transfer procedure was shown to be safe, and technically feasible. Both RRz2- and LNL6-gene-containing peripheral blood mononuclear cells (PBMC) were detected at all time points examined to 4 years. There was concomitant gene construct expression in the absence of the need for ex vivo peripheral blood cell stimulation and there was no evidence of immune elimination of the neoR T-lymphocytes nor of silencing of the Moloney murine leukemia virus long terminal repeat. CONCLUSIONS: The proof of principle results reported here demonstrate safety and feasibility of this type of gene transfer approach. While not specifically tested, T-lymphocytes containing an anti-HIV gene construct may impact on HIV-1 viral load and CD4+ T-lymphocyte count, potentially representing a new therapeutic modality for HIV-1 infection.

Adult↗

PubMed2005

Effect of EBV LMP1 targeted DNAzymes on cell proliferation and apoptosis.

The latent membrane protein (LMP1) encoded by Epstein-Barr virus (EBV) has been suggested to be one of the major oncogenic factors in EBV-mediated carcinogenesis. RNA-cleaving DNA enzymes are catalytic nucleic acids that bind and cleave a target RNA in a highly sequence-specific manner. In this study, we explore the potential of using DNAzymes as a therapeutic approach to EBV-associated carcinomas by targeting the LMP1 gene. In all, 13 different phosphorothioate-modified "10-23" deoxyribozymes (DNAzymes) were designed and synthesized against the LMP1 mRNA and transfected into B95-8 cells, which constitutively express the LMP1. Fluorescence microscopy was used to examine the cellular uptake and distribution in B95-8 cells. As demonstrated in Western blots, three out of 13 deoxyribozymes significantly downregulated the expression of LMP1 in B95-8 cells. These DNAzymes were shown to markedly inhibit B95-8 cell growth compared with a disabled DNAzyme and untreated controls, as determined by an alamarBlue Assay. It was further demonstrated that these DNAzymes arrested the B95-8 cells in G0/G1 using flow cytometry. Interestingly, the active DNAzymes could also downregulate the expression of Bcl-2 gene in treated cells, suggesting a close association between the LMP1 and Bcl-2 genes and their involvement in apoptosis. This was further confirmed with the result that the DNAzymes could induce the release of cytochrome c from mitochondria, which is the hallmark of the apoptosis. The present results suggest that the LMP1 may present a potential target for DNAzymes towards the EBV-associated carcinoma through cell proliferation and apoptosis pathways.

Animals↗

PubMed2004

Homogeneous real-time detection and quantification of nucleic acid amplification using restriction enzyme digestion.

A method for real-time fluorescent detection and quantification of nucleic acid amplification using a restriction endonuclease was developed. In this homogeneous system detection is mediated by a primer containing a reporter and quencher moiety at its 5' terminus separated by a short section of DNA encoding a restriction enzyme recognition sequence. In the single stranded form, the signal from the fluorescent reporter is quenched due to fluorescence resonance energy transfer. However, as the primer becomes incorporated into a double stranded amplicon, a restriction enzyme present in the reaction cleaves the DNA linking the reporter and quencher, allowing unrestricted fluorescence of the reporter. To test this system, a primer specific for the E6 gene of human papilloma virus (HPV) 16 was combined with the cleavable energy transfer label and used to amplify HPV16 positive DNA. In the presence of the thermally stable restriction enzyme BstNI, the reporter system was found to generate a fluorescent signal in proportion to the amount of template DNA. In addition to this direct format, the reporter primer was also used to monitor and quantify the amplification of other sequences. This was accomplished by using primers that contain a tag sequence complementary to the reporter oligonucleotide.

Cell Line, Tumor↗

PubMed2004

Inhibition of human breast carcinoma proliferation, migration, chemoinvasion and solid tumour growth by DNAzymes targeting the zinc finger transcription factor EGR-1.

DNAzymes (synthetic catalytic DNA) have emerged as a new class of nucleic acid-based gene silencing agent. Using DNAzymes targeting the human mRNA of the immediate-early gene and C2H2-class zinc finger transcription factor early growth response-1 (EGR-1), we demonstrate here that EGR-1 plays an indispensable role in breast cancer proliferation, migration, chemoinvasion and xenograft growth in nude mice. DNAzyme inhibition of these tumorigenic processes and EGR-1 protein expression in breast carcinoma cells is sequence-specific and EGR-1 transcription-independent. These agents inhibit breast carcinoma cell migration and chemoinvasion in microchemotaxis chambers and solid tumour growth in athymic nude mice. Thus, DNAzymes targeting specific genes can inhibit multiple key tumorigenic processes in vitro and in vivo and may serve as useful anti-cancer agents.

Animals↗

PubMed2004

Effect of deoxyribozymes targeting c-Jun on solid tumor growth and angiogenesis in rodents.

BACKGROUND: The basic region-leucine zipper protein c-Jun has been linked to cell proliferation, transformation, and apoptosis. However, a direct role for c-Jun in angiogenesis has not been shown. METHODS: We used human microvascular endothelial cells (HMEC-1) transfected with a DNAzyme targeting the c-Jun mRNA (Dz13), related oligonucleotides, or vehicle in in vitro models of microvascular endothelial cell proliferation, migration, chemoinvasion, and tubule formation, a rat model of corneal neovascularization, and a mouse model of solid tumor growth and vascular endothelial growth factor (VEGF)-induced angiogenesis. All statistical tests were two-sided. RESULTS: Compared with mock-transfected cells, HMEC-1 cells transfected with Dz13 expressed less c-Jun protein and possessed lower DNA-binding activity. Dz13 blocked endothelial cell proliferation, migration, chemoinvasion, and tubule formation. Dz13 inhibited the endothelial cell expression and proteolytic activity of MMP-2, a c-Jun-dependent gene. Dz13 inhibited VEGF-induced neovascularization in the rat cornea compared with vehicle control (Dz13 versus vehicle: 4.0 neovessels versus 30.7 neovessels, difference = 26.7 neovessels; P =.004; area occupied by new blood vessels for Dz13 versus vehicle: 0.35 mm2 versus 1.52 mm2, difference = 1.17 mm2; P =.005) as well as solid melanoma growth in mice (Dz13 versus vehicle at 14 days: 108 mm3 versus 283 mm3, difference = 175 mm3; P =.006) with greatly reduced vascular density (Dz13 versus vehicle: 30% versus 100%, difference = 70%; P<.001). CONCLUSION: DNAzymes targeting c-Jun may have therapeutic potential as inhibitors of tumor angiogenesis and growth.

Angiogenesis Inhibitors↗

PubMed2004

Target-site selection for the 10-23 DNAzyme.

The 10-23 DNAzyme is capable of cleaving RNA with high sequence specificity at sites that contain purine-pyrimidine (R-Y) junctions. Although they are abundant in mRNA, many of these potentially cleavable junctions are protected from DNAzyme activity by secondary structure. To optimise the process of target-site selection in long RNA substrates, a multiplex assay was developed for simultaneous comparative analysis of 50 or more different DNAzymes in one reaction. Using this approach, the efficiency of 80 DNAzyme sites within the E6 component of a full-length HPV16 E6/E7 transcript was examined. The activity of molecules selected in this system was then compared in a conventional assay with DNAzymes of intermediate and low performance. This confirmed the results observed in the multiplex reactions, with 10% of DNAzymes inducing substantial cleavage of the long transcript. These DNAzyme-sensitive regions are potentially accessible to other RNA directed agents such as ribozymes or antisense oligonucleotides. Therefore, in addition to finding the most effective DNAzymes for a particular target mRNA, this method may also be applicable to locating accessible sites for other nucleic acid-based gene suppression strategies.

Base Sequence↗

PubMed2004

Nucleic acid sequence analysis using DNAzymes.

The sequence specificity of the "10-23" RNA-cleaving DNA enzyme can be utilized to discriminate between subtle differences in nucleic acid sequence. We examined this potential by comparing the cleavage activity of DNAzymes that target sequences derived from a relatively conserved segment of the L1 gene from different human papillomavirus (HPV) genotypes. DNAzyme activity was found to be highly sensitive to mismatches between its binding domain and substrate sequences containing polymorphisms. Type-specific DNAzyme-cleavable substrates can also be generated by genomic PCR using a chimeric primer containing three bases of RNA. The RNA component enables each amplicon to be cleavable in the presence of its matching DNAzyme. In this format, the specificity of DNAzyme cleavage is defined by Watson-Crick interactions between one substrate-binding domain (arm I) and the polymorphic sequence that is amplified during polymerase chain reaction (PCR). DNAzyme-mediated cleavage of amplicons generated by this method was used to examine the HPV status of genomic DNA derived from Caski cells, which are known to be positive for HPV16. This method is applicable to many types of nucleic acid sequence variation, including single-nucleotide polymorphisms (SNPs).

Base Sequence↗

PubMed2003

Transcription factor Egr-1 supports FGF-dependent angiogenesis during neovascularization and tumor growth.

Current understanding of key transcription factors regulating angiogenesis is limited. Here we show that RNA-cleaving phosphodiester-linked DNA-based enzymes (DNAzymes), targeting a specific motif in the 5' untranslated region of early growth response (Egr-1) mRNA, inhibit Egr-1 protein expression, microvascular endothelial cell replication and migration, and microtubule network formation on basement membrane matrices. Egr-1 DNAzymes blocked angiogenesis in subcutaneous Matrigel plugs in mice, an observation that was independently confirmed by plug analysis in Egr-1-deficient animals, and inhibited MCF-7 human breast carcinoma growth in nude mice. Egr-1 DNAzymes suppressed tumor growth without influencing body weight, wound healing, blood coagulation or other hematological parameters. These agents inhibited endothelial expression of fibroblast growth factor (FGF)-2, a proangiogenic factor downstream of Egr-1, but not that of vascular endothelial growth factor (VEGF). Egr-1 DNAzymes also repressed neovascularization of rat cornea. Thus, microvascular endothelial cell growth, neovascularization, tumor angiogenesis and tumor growth are processes that are critically dependent on Egr-1.

Animals↗

PubMed2003

Optimisation of the 10-23 DNAzyme-substrate pairing interactions enhanced RNA cleavage activity at purine-cytosine target sites.

The 10-23 RNA cleaving DNAzyme has been shown to cleave any purine-pyrimidine (RY) junction under simulated physiological conditions. In this study, we systematically examine the DNAzymes relative activity against different RY combinations in order to determine the hierarchy of substrate core dinucleotide sequence susceptibility. The reactivity of each substrate dinucleotide compared in the same background sequence with the appropriately matched DNAzyme was found to follow the scheme AU = GU >> or = GC >> AC. The relatively poor activity of the DNAzyme against AC and GC containing substrates was found to be improved substantially by modifications to the binding domain which subtly weaken its interaction with the substrate core. The most effective modification resulting in rate enhancement of up to 200-fold, was achieved by substitution of deoxyguanine with deoxyinosine such that the base pair interaction with the RNA substrates core C is reduced from three hydrogen bonds to two. The increased cleavage activity generated by this modification could be important for application of the 10-23 DNAzyme particularly when the target site core is an AC dinucleotide.

2-Aminopurine↗

PubMed2003

E6AP gene suppression and characterization with in vitro selected hammerhead ribozymes.

E6AP was originally identified as the ubiquitin-protein ligase involved in human papillomavirus (HPV) E6-mediated p53 degradation and has since been shown to act as an E3 ubiquitin-protein ligase in the ubiquitination of several other protein substrates. To further define E6AP function at the molecular and cellular levels, a ribozyme-based gene inactivation approach was adopted. A library of hammerhead ribozymes, with randomized arm sequences, was used to screen active molecules along the entire E6AP transcript for ribozyme-cleavable sites. Ligation-anchored PCR was adapted to detect cleavage products, and ribozymes designed to the selected sites were characterized both in vitro and in vivo. Ribozyme-mediated reduction in E6AP expression was found to enhance the apoptotic response of HeLa cells to mitomycin C-induced DNA damage. These findings suggest that E6AP has potential as a drug target, as its suppression can potentiate apoptosis in HPV-positive cells treated with a cytotoxic drug.

Apoptosis↗

PubMed2002

Cellular uptake, distribution, and stability of 10-23 deoxyribozymes.

The cellular uptake, intracellular distribution, and stability of 33-mer deoxyribozyme oligonucleotides (DNAzymes) were examined in several cell lines. PAGE analysis revealed that there was a weak association between the DNAzyme and DOTAP or Superfect transfection reagents at charge ratios that were minimally toxic to cultured cells. Cellular uptake was analyzed by cell fractionation of radiolabeled DNAzyme, by FACS, and by fluorescent microscopic analysis of FITC-labeled and TAMRA-labeled DNAzyme. Altering DNAzyme size and chemistry did not significantly affect uptake into cells. Inspection of paraformaldehyde-fixed cells by fluorescence microscopy revealed that DNAzyme was distributed primarily in punctate structures surrounding the nucleus and that substantial delivery to the nucleus was not observed up to 24 hours after initiation of transfection. Incubation in human serum or plasma demonstrated that a 3'-inversion modification greatly increased DNAzyme stability (t(1/2) approximately 22 hours) in comparison to the unmodified form (t(1/2) approximately 70 minute). The 3'-inversion-modified DNAzymes remained stable during cellular uptake, and catalytically active oligonucleotide could be extracted from the cells 24 hours posttransfection. In smooth muscle cell proliferation assay, the modified DNAzyme targeting the c-myc gene showed a much stronger inhibitory effect than did the unmodified version. The present study demonstrates that DNAzymes with a 3'-inversion are readily delivered into cultured cells and are functionally stable for several hours in serum and within cells.

Base Sequence↗