ABSTRACT
<p><b>OBJECTIVE</b>To specifically deliver the therapeutic gene to cancer cells and construct target retroviral vectors by inserting the single-chain variable antibody fragment into the retroviral envelope.</p><p><b>METHODS</b>Single-chain antibody expression vector pET -20bScfv was constructed. Binding activity of the genetically engineered single-chain variable antibody fragment was verified by enzyme-linked immunosorbent assay (ELISA) and Western blot. At the same time, by means of polymerase chain reaction (PCR)-directed mutagenesis, the appropriate cloning site EcoT22/Sal was generated at the N-terminus of receptor-binding SU domain in the MoMLV env polypeptide. Then the single- chain antibody gene, encoding a functional antibody, was inserted into the cloning site. The Scfv-env fusion gene fragment was subcloned into CMV expression vector. The Lac-Z retrovirus that co-displayed the Scfv-env chimeric protein and wild-type env protein was produced by transfection of Psi 2 cells with retroviral plasmid and the fusion gene expressing plasmid. To confirm the specificity of the recombinant retrovirus, infection assays and competitive inhibition assays were performed.</p><p><b>RESULTS</b>The results of ELISA and Western blot showed that the genetically engineered single-chain variable antibody fragment could bind to the SHG44 cells surface membrane antigen. Virus-binding assay, viral infection and competitive inhibition assays confirmed that the harvested virus efficiently bound to and infected SHG44 cancer cells expressing the relative membrane antigen specially via the recognition of the target antigen.</p><p><b>CONCLUSION</b>These results imply that insertion of Scfv into the retroviral envelope can specifically deliver the interested gene into specific antigen-producing cancer cells.</p>
Subject(s)
Humans , Gene Transfer Techniques , Genetic Therapy , Genetic Vectors , HeLa Cells , Immunoglobulin Fragments , Genetics , Neoplasms , Therapeutics , Retroviridae , GeneticsABSTRACT
Purpose To study the reversal effect on multidrug resistance (MDR) by TNF-α gene combined with verapamil (VRP) or tamoxifen (TAM). Methods By using recombinant retrovirus vector, TNF-α gene was transfected into multidrug-resistant human breast cancer cell line MCF7/ADR. The TNF-α secreting cell clone MCF7/ADR-TNF was obtained by G418 screening. The integrating and secreting of TNF-α were analyzed by PCR and ELISA. MTT assay and formula"I = d/D1 + d/D2" were used to evaluate the reversal effect of multidmg resistance with TNF-α gene combined with verapamil or tamoxifen. ResultsThe level of TNF-α secreted by MCF7/ADR-TNF was 1 737 pg/ml (106cells/48 h). Compared with control,the resistance to ADR of MCF7/ADR-TNF was reversed by 1.6 times. The reversal effect produced by combination of TNF-α gene and VRP was antagonistic. The combination of TNF-α gene and TAM produced synergic effect (interaction index I = 0.64). ConclusionsTNF-α gene combined with TAM has synergic effect on reversing MDR.
ABSTRACT
In order to use retroviral-mediated gene transfer technology in clinical application, retroviral vector must be of high titer and free of detectable replication-competent retroviruses (RCR). The aim of this study was to optimize methods of defective retroviral vector production. Study was conducted using a LXSN vector inserted with human tumor necrosis factor-? gene and an amphotropic retrovirus packaging cell line-PA317. The results indicated that viral titer was influenced by volume of medium and concentration of fetal calf serum. Inactivation of retroviral vector was greater at 37癈 than at 32癈. In experiment of transfection of PA317 and transduction of 3T3, integration of retroviral vector into genome of packaging cells and target cells, and free of RCR were detected by polymerase chain reaction analysis. Viral vector with high titer and free of RCR is able to use in clinical trial
ABSTRACT
We selected defective retrovirus LXSN approved by the recombinant DNA Advisory Comittee, NIH, USA as a vector to study the transduction of human TIL with TNF gene, on the basis of which, we engaged in the clinical application. The results showed that the logarithmic growth phase was the optimal transducing time when the TIL were isolated and cultured from 9 to 25 days. It also showed that the transduction efficiency was correlated to the MOI value, the more MOI value, the more TIL being transduced. Successful gene inserted and expressing were confirmed by the polymerase chain reaction and the L929 cell test. The clinical trial of treatment for metastastic hepatic cancer suggested that the better therapeutic effectiveness and less side effect depend on the specificity of the TIL, the proper infusion pathway and the dose of the transduced TIL.
ABSTRACT
Phenotypic markers of leukemic cells from 29 children with acute leukemia were examined. Of these cases, six were negative for myeloperoxidase and did not react with lineage-associated or mature lineage-associated monoclonal antibodies. Then, we analyzed the configuration of both immunoglobulin (heavy chain and kappa chain) and T-cell receptor (?, ?, ?) genes in these six cases. All cases had rearrangement of IgH were suggestive of B-lymphoid origin of these leukemic cells. Two cases without CD10 antigen had no rearrangement of kappa chain, one with retention of the germline configuration of TCR ?, ?. ?, the other with retention of the germline of TCR ? gene. In the cases with CD10 antigen, two cases showed kappa chain deletion, the alteration of TCR genes (rearrangements or deletions )was shown to occur frequently. These findings may implicate that the gene rearrange ments forming functional IgH gene in leukemic cells with CD10 antigen, induce the alteration of IgL and TCR genes.