Conditionally replicating adenovirus SG500-expressed mutant Dm-dNK gene for breast cancer therapy
1Department of Breast Surgery, Fourth Affiliated Hospital, China Medical University, Shenyang, PR China.
Abstract:
The purpose of this analysis was to investigate the enzyme activity and specificity of using adenovirus-mediated Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) mutants in combination with gemcitabine. Compared with herpes simplex type 1 thymidine kinase (HSV-TK) and other known dNKs, this Dm-dNK enzyme has a broader substrate specificity and a higher catalytic rate. We created the Dm-dNK mutants (dNKmu) by site-directed mutagenesis at the sites of 244E, 245S, 251S and 252R, with the last 10 amino acids in the amino acid sequence randomly mutated. We evaluated the enzyme activity and substrate specificity. The engineered enzymes showed a relative increase in phosphorylation in the nucleoside analogs of BVDU ((E)-5‑(2-Bromovinyl)-2'-deoxyuridine) or gemcitabine (DFDC, 2',2'-difluoro-deoxycytidine) compared with the wild-type enzyme. The dNKmu enzymes were expressed in the breast cancer cell lines MDA-MB-231 (ER-) and MCF7 (ER+). In studying the sensitivity of the cell lines to DFDC, conditionally replicative adenovirus (CRAd) SG500-dNKmu showed higher expression and enzymatic activity than the replication-defective adenovirus SG500 in cancer cells, but with less cytotoxicity to cancer cells than that of SG500. Our data suggest that the triple phosphorylated DFDC catalyzed by dNKmu inhibited the replication of adenovirus with a simultaneous positive therapeutic effect to cancer cells. Therefore, concomitant use of the SG500‑dNKmu and DFDC could be a novel targeted strategy in suicide gene therapy with safe control of excessive virus replication.
Insights
Engineered Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) mutants show enhanced gemcitabine phosphorylation for targeted cancer therapy. This approach offers a novel suicide gene therapy strategy with controlled virus replication and therapeutic benefits.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Gene Therapy
Background:
- Adenovirus-mediated gene therapy is a promising cancer treatment strategy.
- Deoxyribonucleoside kinases (dNKs) play a crucial role in nucleoside analog activation.
- Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) exhibits unique enzymatic properties.
Purpose of the Study:
- To investigate the enzyme activity and specificity of Dm-dNK mutants for gemcitabine phosphorylation.
- To evaluate the therapeutic potential of Dm-dNK mutants in combination with gemcitabine in breast cancer cell lines.
- To assess the safety and efficacy of adenovirus-mediated Dm-dNK suicide gene therapy.
Main Methods:
- Site-directed mutagenesis was used to create Dm-dNK mutants (dNKmu).
- Enzyme activity and substrate specificity were evaluated using nucleoside analogs like gemcitabine (DFDC).
- Conditionally replicative adenovirus (CRAd) SG500-dNKmu was engineered and tested in MDA-MB-231 and MCF7 breast cancer cells.
Main Results:
- Engineered Dm-dNK mutants demonstrated increased phosphorylation of gemcitabine compared to wild-type.
- CRAd SG500-dNKmu showed higher expression and enzymatic activity in cancer cells.
- Triple phosphorylated DFDC inhibited adenovirus replication, leading to a therapeutic effect on cancer cells.
Conclusions:
- Dm-dNK mutants possess broader substrate specificity and higher catalytic rates, making them suitable for gene therapy.
- Concomitant use of SG500-dNKmu and gemcitabine represents a novel suicide gene therapy strategy.
- This approach offers targeted cancer therapy with safe control of excessive virus replication.
