Tumor cell-specific gene transfer with retroviral vectors displaying single-chain antibody.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
These results imply that insertion of Scfv into the retroviral envelope can specifically deliver the interested gene into specific antigen-producing cancer cells.
Tang
Yucheng
Y
Department of Biochemistry and Molecular Biology, the Research Center for Human Gene Therapy, Shanghai Second Medical University, Shanghai 200025, China.
Li
Yu
Y
Qian
Guanxiang
G
D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics*; D009369:Neoplasms / Q000628:therapy*; D012190:Retroviridae / Q000235:genetics*
D007128:Immunoglobulin Fragments; C055966:immunoglobulin Fv