Group Ⅱ introns and the application in biotechnology: a review / 生物工程学报

Group Ⅱ introns are self-splicing ribozymes, which insert directly into target sites in DNA with high frequency through "retrohoming". They specifically and efficiently recognize and splice DNA target sites, endowing themselves with great potential in genetic engineering. This paper reviewed the gene targeting principle of group Ⅱ introns and the application in microbial genetic modification, and then analyzed the limitations of them in multi-functional gene editing and eukaryotes based on the "retrohoming" characteristics and the dependence on high Mg2+ concentration. Finally, we dissected the potential of group Ⅱ introns in the development of novel gene editing tools based on our previous research outcome and the structural characteristics of the introns, hoping to provide a reference for the application of group Ⅱ introns in biotechnology.

Ribozyme riboswitch based gene expression regulation systems for gene therapy applications: progress and challenges / 药学学报

Robust and efficient control of therapeutic gene expression is needed for timing and dosing of gene therapy drugs in clinical applications. Ribozyme riboswitch provides a promising building block for ligand-controlled gene-regulatory system, based on its property that exhibits tunable gene regulation, design modularity, and target specificity. Ribozyme riboswitch can be used in various gene delivery vectors. In recent years, there have been breakthroughs in extending ribozyme riboswitch's application from gene-expression control to cellular function and fate control. High throughput screening platforms were established, that allow not only rapid optimization of ribozyme riboswitch in a microbial host, but also straightforward transfer of selected devices exhibiting desired activities to mammalian cell lines in a predictable manner. Mathematical models were employed successfully to explore the performance of ribozyme riboswitch quantitively and its rational design predictably. However, to progress toward gene therapy relevant applications, both precision rational design of regulatory circuits and the biocompatibility of regulatory ligand are still of crucial importance.

Construction of a full-genome HCV replicon with self-cleaving double ribozyme sequences and characterization in vitro and in vivo / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct a full-genome hepatitis C virus (HCV) replicon that will allow for direct initiation of replication and generation of infectious viral particles in an in vitro and in vivo cell system.</p><p><b>METHODS</b>Self-cleaving ribozyme sequences were added to each side of the HCV cDNA clone JFH1 and the replication-deficient clone JFH1/GND, then inserted into the pcDNA3.1 vector downstream of the CMV promoter. The resultant recombinant plasmids, pcDNA3.1-RZ-JFH1 and pcDNA3.1-RZ-JFH1/GND, were tested for activity in vitro and in vivo by transiently transfecting into Huh7.5 cells (5 mug/100 mm culture dish) and injecting by high-pressure tail vein injection into Kunming mice (10 - 30 mug/mouse). Quantitative reverse transcription-PCR, immunofluorescence, immunohistochemistry, and serological testing were performed to determine the replication ability and assess the properties of the recombinant plasmids in the two systems.</p><p><b>RESULTS</b>HCV RNA (1 - 3 * 10(6) copies/ml) was detected in the supernatant of transfected Huh7.5 cells up to 16 weeks after transfection. In addition, the viral particles from the supernatant were able to infect nave Huh7.5 cells. However, only transient viremia was achieved upon tail vein injection of the plasmid, and no HCV antigen-positive cells were detected by immunohistochemistry nor HCV-specific antibodies by serological testing.</p><p><b>CONCLUSION</b>The constructed HCV replicon was capable of stable expression in cultured cells and of efficiently generating infectious viral particles in the in vitro system over a long period. However, the HCV replicon did not show infective characteristics in an in vivo mouse system. The full-length HCV replicon may represent a useful tool for in vitro study of HCV pathological mechanisms, possibly including anti-HCV drug screening.</p>

Construction of an engineered M1GS-HCV/C141 ribozyme and determination of its antiviral activity in vitro / 生物工程学报

Hepatitis C virus (HCV), one of the major pathogens of viral hepatitis, causes significant hazards in humans. Interferon treatment in combination with ribavirin is used as the first line clinical treatment for HCV infection. However, good response to this treatment has only been observed in few patients and repeated recurrence has also been reported frequently. Therefore, new antiviral agents and therapies are in urgent demand. Here, we report a newly constructed Escherichia coli RNase P based M1GS ribozyme that can specifically and efficiently target the core gene of HCV. The guide sequence (GS) of this M1IGS was designed according to the sequence of the core coding region of HCV genome. The GS was then covalently linked to the 3' terminus of M1 RNA, the catalytic subunit of RNase P from Escherichia coli. The specification of this sequence-specific ribozyme, M1GS, was then examined using an in vitro cleavage assay. The cytotoxicity and its activity in inhibition of HCV gene expression and viral proliferation were further studied in vivo. Our results show that the reconstructed M1GS ribozyme displayed obvious catalytic activity in cleaving target mRNAs fragment in vitro. Notable reduction in the expression of HCV core protein and a 1 000-fold reduction in viral growth were also observed in cultured HCV infected Huh7.5.1 cells expressing the functional M1GS ribozyme. This study demonstrated a direct evidence for the antiviral activity of the customized M1GS-HCV/C141 ribozyme, and thus provided a promising new strategy for clinical treatment of HCV infection.

Engineering and screening of artificial riboswitch as a novel gene control element / 生物工程学报

Various artificial riboswitches have been constructed by utilization of designed aptamers or by modification of natural riboswitch systems, because they can regulate gene expression in a highly efficient, precise and fast way, and promise to supply simple cis-acting, modular, and non-immunogenic system for use in future gene therapy applications. In this review, we present an overview of currently available technologies to design and select engineered riboswitches, and discuss some possible technologies that would allow them highly responsive to non-natural ligands, and dynamic control of gene expression in mammalian cells. Though how to bring custom-designed riboswitches as a novel and versatile tool box to gene control system is still a great challenge, the combination of structure-activity relationship information, computer based molecular design, in vitro selection, and high-through screening will serve as powerful tools for further development of riboswitch based gene regulatory systems.

Terapia génica: los ácidos nucleicos como fármacos: mecanismos de acción y vías de entrega a la célula / Gene therapy: nucleic acids as drugs: action mechanisms and delivery into the cell

La terapia génica involucra la transferencia de material genético a una célula para conseguir un beneficio terapéutico, por lo que ofrece una nueva opción para el tratamiento de muchas patologías. En este artículo se presentan algunos de los nuevos fármacos a base de ácidos nucleicos, como plásmidos, aptámeros, oligonucleótidos, ribozimas y pequeños ácidos ribonucleicos de interferencia. Se comenta el mecanismo y nivel de acción de estos agentes terapéuticos y se discuten sus varias vías de administración, como liposomas, polímeros catiónicos, transferencia directa de ácidos nucleicos y vectores virales.

Gene therapy involves the transference of new genetic material to the cell in order to obtain a therapeutic benefit, offering a new option for the treatment of various diseases. In this article, some of these nucleic acid-based drugs, such asplasmids, aptamers, oligonucleotides, ribozymes and small interfering ribonucleic acid, are presented. Their mechanism and level of action iscommented and several delivery systems, such as liposomes, cationic polymers, direct nucleic acid transfer and viral vectors, are also discussed.

Construction of HCV-producing cell model based on self-cleaving ribozyme / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To construct a stable HCV-producing cell model for anti-HCV drug research.</p><p><b>METHODS</b>The HCV-ribozyme recombinant plasmid pJFH1-Rbz was constructed to generate the exact 5' and 3' ends of HCV genomic RNA by placing two self-cleaving ribozymes at both ends of the HCV JFH-1 cDNA. The plasmid was then transfected into HepG2 cells and the resultant clones were screened with G418. Subsequently, immunofluorescence and Western blot were performed to detect the expression of HCV core protein, HCV RNA level was quantitated by TaqMan real-time PCR method and HCV particles was detected by electron microscopy.</p><p><b>RESULTS</b>HCV core protein was detected in the screened cell clone, and the level of HCV RNA was up to 1000,0000 copies/ml in the culture medium. Electron microscopy showed the viral particles in the culture suspension were approximately 55 nm in diameter. IFN-treating experiment demonstrated that the HCV RNA level decreased with the increasing concentration of IFN alpha.</p><p><b>CONCLUSION</b>We constructed a stable HCV-producing cell model which can be used for anti-HCV drug research.</p>

Effects of short hairpin RNA targeting epidermal growth factor receptor on the radiosensitivity of human nasopharyngeal carcinoma xenografts in nude mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of the vector carrying short hairpin RNA targeting epidermal growth factor receptor (shRNA-EGFR) on the radiosensitivity of human nasopharyngeal carcinoma xenografts in nude mice.</p><p><b>METHODS</b>shRNA-EGFR was transfected into human nasopharyngeal carcinoma cell line CNE1 via Lipofectamine 2000. The transfected cells were collected for quantitative RT-PCR detection of the expression level of EGFR mRNA. Western blotting was used to examine the expression of EGFR protein. CNE1 cells were inoculated into nude mice and the tumor volume was measured every 2 days. shRNA-EGFR was intratumorally injected in the mice, and 16 days after radiotherapy, the mice were sacrificed and tumors examined for radiosensitivity.</p><p><b>RESULTS</b>shRNA-EGFR was effectively delivered via Lipofectamine 2000 into CNE cells to result in a significant downregulation of EGFR mRNA and protein expressions (P<0.05). A significant difference was noted in the tumor volume and weight in the tumor-bearing nude mice between shRNA-EGFR plus radiotherapy group and the control, exclusive radiotherapy and shRNA-EGFR groups (P<0.05).</p><p><b>CONCLUSION</b>shRNA-EGFR combined with radiotherapy can effectively inhibit the growth of nasopharyngeal carcinoma in nude mice. shRNA-EGFR can enhance sensitivity of nasopharyngeal carcinoma to radiotherapy.</p>

Progress in the research of therapeutic enzyme / 生物工程学报

With the development of the research on biotechnology and modern pharmacy, the application of enzyme drugs have grown rapidly and enzyme drugs have become an important branch of biopharmaceutics. In this article, some new varieties of therapeutic enzymes, enzyme targets, mechanisms and new technologies of application in therapeutic enzymes were reviewed, and the direction of development of therapeutic enzymes were discussed.

High-level transcription of human mitochondrial tRNA(Trp) based on ribozyme and characterization on its aminoacylation activity / 生物工程学报

In vitro transcription systems with T7 RNA polymerase (T7 RNAP) were widely used in preparation of RNA because of their simplicity and high efficiency. The transcripts would have additional 5' sequence since T7 promoter spans the transcription start site, while deletion of the transcription start site would severely reduce the T7 RNAP transcriptional activity. We successfully developed an in vitro transcription by combining of T7 RNAP high efficient transcription system and highly specific self-splicing technology of ribozymes, in this system, ribozyme self-splices at the designed specific site and releases the aim RNA without affecting transcription efficiency of T7 RNAP, the aminoacylation activity of human mitochondrial tRNA(Trp) (HmtRNA(Trp) (UCA)) is 113.6 pmol/microg. This method with its high efficiency on transcription and good repeatability is very suitable for preparation of accurate RNA in large scale.

RNA Mapping of Mutant Myotonic Dystrophy Protein Kinase 3'-Untranslated Region Transcripts

Myotonic dystrophy type 1 (DM1), which is a dominantly inherited neurodegenerative disorder, results from a CTG trinucleotide repeat expansion in the 3'-untranslated region (3'-UTR) of the myotonic dystrophy protein kinase (DMPK) gene. Retention of mutant DMPK (mDMPK) transcripts in the nuclei of affected cells has been known to be the main cause of pathogenesis of the disease. Thus, reducing the RNA toxicity through elimination of the mutant RNA has been suggested as one therapeutic strategy against DM1. In this study, we suggested RNA replacement with a trans -splicing ribozyme as an alternate genetic therapeutic approach for amelioration of DM1. To this end, we identified the regions of mDMPK 3'-UTR RNA that were accessible to ribozymes by using an RNA mapping strategy based on a trans - splicing ribozyme library. We found that particularly accessible sites were present not only upstream but also downstream of the expanded repeat sequence. Repair or replacement of the mDMPK transcript with the specific ribozyme will be useful for DM1 treatment through reduction of toxic mutant transcripts and simultaneously restore wild-type DMPK or release nucleus-entrapped mDMPK transcripts to the cytoplasm.

Progress of antisense technology applied in metabolic regulation of bacteria / 生物工程学报

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.

An RNA Mapping Strategy to Identify Ribozyme-Accessible Sites on the Catalytic Subunit of Mouse Telomerase

Telomerase reverse transcriptase (TERT) is an enzymatic ribonucleoprotein that prolongs the replicative life span of cells by maintaining protective structures at the ends of eukaryotic chromosomes. Telomerase activity is highly up-regulated in 85-90% of human cancers, and is predominately regulated by hTERT expression. In contrast, most normal somatic tissues in humans express low or undetectable levels of telomerase activity. This expression profile identifies TERT as a potential anticancer target. By using an RNA mapping strategy based on a trans-splicing ribozyme library, we identified the regions of mouse TERT (mTERT) RNA that were accessible to ribozymes. We found that particularly accessible sites were present downstream of the AUG start codon. This mTERTspecific ribozyme will be useful for validation of the RNA replacement as cancer gene therapy approach in mouse model with syngeneic tumors.

Experimental study on the role of VEGF autocrine loop in K562 leukemia cells / 中华血液学杂志

<p><b>OBJECTIVE</b>lo investigate the effects of anti-VEGF antibody and anti-VEGF hairpin ribozyme gene on the proliferation, apoptosis and related gene expression of the leukemia K562 cells and the possible molecular mechanisms.</p><p><b>METHODS</b>K562 cells were cultured in different concentrations of anti-VEGF antibody. The recombinant eukaryotic expression plasmid (pcDNA3-RZ) containing anti-VEGF hairpin ribozyme gene and the vector-alone were introduced into K562 cells by lipofectamine mediation. Cell proliferative capacity was determined by MTT, colony formation assay and cells cycles analysis. Cell apoptosis was assayed by DNA ladder and Annexin V -FITC/PI flow cytometry.</p><p><b>RESULTS</b>The anti-VEGF antibody was able to inhibit growth and induce apoptosis of K562 cells in a dose-dependent manner. Exposure to anti-VEGF antibody at 0. 165 microg/ml for 72 hours, the cells exhibited typical DNA ladders. The apoptosis rate peaked at antibody concentration of 0. 825 microg/ml. RT-PCR showed a decrease of MRP and TOPO II expression but a relative constant expression of GST. The introduction of exogenous anti-VEGF ribozyme gene resulted in a decrease of the proliferative capacity and colony forming efficiency from (30.5 +/- 3.3) % in control group to (16.3 +/- 2.8) % in K562/RZ group, higher G1 and lower S ratio in cell cycle distribution in comparison with the control groups. Typical DNA fragmentation and higher Annexin V + ratio occurred in K562/RZ cells after treated with 0.5 micromot/L of As2O3 for 3 days, the apoptosis rate increased from 13.4% in control group to 31. 5% in As2O3 treated group.</p><p><b>CONCLUSION</b>Anti-VEGF antibody can inhibit growth, induce apoptosis and down-regulate the expression of MRP, TOPO II genes of K562 cells in vitro. Transfection with anti-VEGF ribozyme gene can inhibit the proliferation of the cells by delaying the progression G1 into S phase in cell cycles and induce cell apoptosis by down-regulating VEGF gene expression.</p>

Gene expression responses in vivo by human telomerase reverse transcriptase (hTERT)-targeting trans-splicing ribozyme

A trans-splicing ribozyme which can specifically reprogram human telomerase reverse transcriptase (hTERT) RNA was previously suggested as a useful agent for tumor-targeted gene therapy. In this study, we evaluated in vivo function of the hTERT-targeting trans-splicing ribozymes by employing the molecular analysis of expression level of genes affected by the ribozyme delivery into peritoneal carcinomatosis mice model. To this effect, we constructed adenoviral vector encoding the specific ribozyme. Noticeably, more than four-fold reduction in the level of hTERT RNA was observed in tumor nodules by the systemic infection of the ribozyme-encoding virus. Such hTERT RNA knockdown in vivo induced changes in the global gene expression profile, including the suppression of specific genes associated with anti-apoptosis including bcl2, and genes for angiogenesis and metastasis. In addition, specific trans-splicing reaction with the targeted hTERT RNA took place in the tumors established as peritoneal carcinomatosis in mice by systemic delivery of the ribozyme. In conclusion, this study demonstrates that an hTERT-specific RNA replacement approach using trans-splicing ribozyme represents a potential modality to treat cancer.

The down regulation of VEGF gene expression may link to change the expression profile of genes in leukemia cell line K562 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the potential effects of anti-VEGF hairpin ribozyme gene to gene expression profiles in leukemia cell line K562.</p><p><b>METHODS</b>The lipofectamine mediation was used to transfect the recombinant eukaryotic expression plasmid (pcDNA3-RZ) containing anti-VEGF hairpin ribozyme gene and the non-recombinant vector as control into K562 cells. And the positive clones were screened by G418. Ribozyme gene in K562 cells was confirmed by PCR. Fluorescent real time reverse transcription-PCR(RT-PCR) and Western blotting were employed to detect the expression of VEGF mRNA and protein in leukemia cells. cDNA microarray was used to explore the alteration of gene expression profiles when decreasing VEGF gene expression in leukemia cells. Expression of PCNA and GSN genes were verified by semi-quantitative RT-PCR.</p><p><b>RESULTS</b>The pcDNA3-RZ and pcDNA3 had been transfected into the human leukemia cell line K562 and positive clones been screened by G418. Stable expression of the ribozyme gene in K562 cells was confirmed by PCR. The level of VEGF mRNA and protein decreased dramatically in K562-RZ cells when compared with K562 or K562-PC (K562 cell transfected with empty vector) cells. The gene expression profiles were changed by transfection of anti-VEGF hairpin ribozyme gene into K562 cells. Among 4096 gene clones on the microarray, 191 (4.86%) genes were detected to have the marked changes with 104 down-regulated and 87 up-regulated, that were functionally related to cell cycle progression, gene replication, metabolism, cell apoptosis, cell signal transduction, and oncogenes etc. An increased expression of GSN gene and a decreased expression of PCNA gene in K562/RZ cells have been detected by RT-PCR.</p><p><b>CONCLUSION</b>Down-regulation of VEGF gene by introducing anti-VEGF hairpin ribozyme gene can alter the gene expression profiles in K562 cells, leading to change of cell growth, differentiation and apoptosis in K562/RZ cells.</p>

Inhibition of K562 cell growth and tumor angiogenesis in nude mice by transfection of anti-VEGF hairpin ribozyme gene into the cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of anti-VEGF hairpin ribozyme gene on the tumor cell growth and tumor angiogenesis in nude mice.</p><p><b>METHODS</b>The recombinant eukaryotic expression plasmid pcDNA-RZ containing anti-VEGF hairpin ribozyme gene and the empty vector plasmid pcDNA were introduced separately into K562 cells by lipofectamine mediation and positive clones were screened by G418. Ribozyme gene in K562 cells was confirmed by PCR. Fluorescent real time RT-PCR and Western blot were used to detect the expression of VEGF mRNA and protein in the leukemia cells. The tumorigenicity of transfected K562 cells were transplanted in nude mice and tumor microvascular density (MVD) were observed by morphology and vWF immunohistochemistry stain.</p><p><b>RESULTS</b>Stable expression of the ribozyme gene in K562 cells was confirmed by PCR. The level of VEGF mRNA and protein decreased dramatically in K562/RZ cells when compared with K562 or K562/PC (K562 cells transfected with empty vector) cells. The tumor volumes were (4.43 +/- 0.87), (3.96 +/- 0.94), (2.24 +/- 0.56) cm3; tumor weight was (4.43 +/- 0.87), (3.96 +/- 0.94), (2.24 +/- 0.56)g; and tumor microvascular density was 4.70 +/- 1.25, 4.67 +/- 1.31, 1.80 +/- 1.55 in K562, K562/PC and K562/RZ cell groups, respectively.</p><p><b>CONCLUSION</b>Transfection with anti-VEGF ribozyme gene can inhibit tumor growth and vessel formation by down-regulating the VEGF gene expression in K562 cells.</p>

Inhibition of HCV replication by HCV specific U1 small nuclear RNA chimeric ribozyme in vivo / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To determine the advantage of U1 small nuclear RNA as a ribozyme vector (U1-Rz) to inhibit HCV replication in vivo.</p><p><b>METHODS</b>The 3rd stem-loop was substituted by HCV core specific ribozyme to construct an U1-Rz eucaryotic expression plasmid. Then it was co-transfected with pCMV/T7-NCRC Delta-luc into Huh7 cell line mediated by lipofectin. The cell lysis supernatant was subjected to Western blot and lumenometer to determine the luciferase levels.</p><p><b>RESULTS</b>A U1 snRNA chimeric ribozyme was constructed successfully. Both Rz and U1-Rz inhibited luciferase expression in Huh7 by 48.64% and 87.46%, respectively.</p><p><b>CONCLUSION</b>Rz has more efficacy in cells when using U1 snRNA delivery system. U1 can be an efficient vector for HCV specific ribozyme.</p>

Hammerhead ribozyme-mediated cleavage of transforming growth factor beta1 RNA in a cell-free system and in hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To identify the activity of hammerhead ribozyme against transforming growth factor beta1 (TGFbeta1) in a cell-free system and in activated hepatic stellate cells (HSCs).</p><p><b>METHODS</b>The ribozyme against TGFb1 was designed with computer software. The transcripts of ribozyme, disabled ribozyme and target RNAs were prepared using the RiboMAX large scale RNA production system. The in vitro cleavage reactions were performed through incubation of 32P-labeled target RNAs with ribozyme or disabled ribozyme in different conditions. The eukaryotic expression vector encoding ribozyme and disabled ribozyme were constructed, and then transfected into HSC-T6 cells which exhibited characteristics of activated HSCs. The intracellular activity of the ribozyme was determined by detecting the ribozyme, disabled ribozyme and the TGFbeta1 expression.</p><p><b>RESULTS</b>The ribozyme cleaved target RNAs into anticipated products effectively. As expected, the disabled ribozyme possessed no cleavage activity in vitro. Further study demonstrated that the ribozyme expressed efficiently and inhibited TGFbeta1 expression in activated HSCs, while the disabled ribozyme displayed only a slight effect on TGFbeta1 expression.</p><p><b>CONCLUSION</b>The ribozyme with perfect cleavage activity in the cell-free system used inhibited TGFbeta1 expression effectively in activated HSCs. This ribozyme can provide a potential therapeutic approach for liver fibrosis.</p>

Depressing the immune escape of acute myelomonocytic leukemia via an anti-Fas ribozyme / 中国实验血液学杂志

In order to investigate the inhibition role of anti-Fas hammerhead ribozyme on Fas expression and Fas-mediated apoptosis in CTLL-2 cells (mouse CTL cell line), and to explore a new way for enhancing the ability of T cells against Leukemia in donor lymphocytes infusion, CTLL-2 cells were transfected with pEGFP-RZ596 and pEGFPC1 (mock-transfected) via electroporation. Fas expression on CTLL-2 cells was detected by RT-PCR and Western blot. The killing effect of CTL against WEHI-3 (mouse acute myelomonocytic leukemia cell line) highly expressing FasL in vitro was detected by MTT assay. The caspase-3 proteolytic activity and the apoptosis rate of CTLL-2 cells were detected by means of BD AproAlert Caspase-3 Colorimetric kit and FITC labeled Annexin-V apoptosis detecting kit respectively. The results showed that the anti-Fas ribozyme could be successfully introduced into mouse CTLL-2 cells; Fas expression on the surface of cells transfected with the ribozyme was obviously decreased, in comparison with control and mock-transfected cells; after cocultured with WEHI-3 cells, the viability of CTLL-2 cells transfeced with the ribozyme was significantly increased, as compared with other two groups; caspase-3 activity and apoptosis rate of the ribozyme-transfeced cells were significantly decreased, the killing effect of CTLL-2 transfected with the ribozyme was stronger than that of other groups. It is concluded that anti-Fas ribozyme can remarkably decrease Fas expression on CTLL-2 cells, so as to avoid Fas-mediated apoptosis by Fas ligand on WEHI-3 cells, and to enhance their killing activity against WEHI-3 cells, as a result, the immune escape of acute myelomonocytic leukemia was depressed.