Scientists have developed a fusion protein-based system for effectively delivering small interfering RNAs to cancer. The technology developed by researchers at Sun Yat-sen Memorial Hospital’s Breast Tumor Center in China hinges on a fusion protein comprising single-chain fragmented antibodies (ScFvs) linked to peptides as a carrier for delivering Polo-like kinase 1 (PLK1) siRNAs into Her2+ tumors. Tests showed that the delivery approach led to the suppression of Her2+ breast cancer cell lines in vitro. In vivo studies showed that intravenous administration of the siRNA-carrier constructs slowed cancer growth, reduced metastasis, and prolonged survival in experimental mice carrying primary human tumors.
Erwei Song, M.D., and colleagues describe their technology and experimental results in Science Translational Medicine in a paper titled “Targeted Delivery of PLK1-siRNA by ScFv Suppresses Her2+ Breast Cancer Growth and Metastasis.”
PLK1 is a promising target for a range of cancers including Her2+ breast tumors, and its inhibition has been shown to lead to cell cycle arrest, apoptosis, and mitotic catastrophe in cancer cells. Prior studies have also indicated that suppressing PLK1 expression using siRNAS may be a promising therapeutic strategy, but problems associated with efficient and targeted delivery of siRNAs remains a major hurdle, the investigators note.
The Chinese team has previously demonstrated that a fusion protein comprising anti-Her2 ScFv and a protamine peptide (FP-5) was capable of knocking down gene expression specifically in Her2+ cells. Their latest work was designed to evaluate use of Her2-ScFv-mediated intravenous delivery of a PLK1-siRNA for inhibiting tumor growth and metastasis of Her2+ breast cancer both in vitro and in vivo.
The F5-P fusion protein comprises an anti-Her2 single-chain antibody fragment linked at its C terminus to a protamine peptide followed by a His6 tag, which bound in solution to the siRNA cargo. Initial tests using fluorescently labelled constructs confirmed that F5-P could efficiently deliver siRNA into Her2+ cells but not Her2- cells, including those isolated from surgically removed breast tumors. Importantly, while naked siRNAs were rapidly excreted, siRNA stabilized and protected by the protamine peptide were delivered directly to the cancer cells and were still detectable 72 hours later.
Encouragingly, analyses of Her2+ cell lines and Her2+ primary breast cancer cells in vitro showed that treatment using the fusion protein-siRNA complex led to reductions in levels of both PLK1 mRNA and protein levels. This was accompanied by significantly lower levels of cell proliferation and colony formation, and increased apoptosis after 72 hours. Equivalent results were obtained using a different PLK1 siRNA complexed with F5-P, “suggesting that the antitumor effect was due to PLK1 knockdown,” the authors note. The correlation between tumor repression and PLK1 knockdown was confirmed through studies in cells expressing an siRNA-insenstiive PLK1, which were resistant to the active siRNA.
The investigators then tested intravenous injections of the F5-P fusion protein-delivered PLK1 siRNA in mice bearing mammary xenografts of breast cancer cell lines or primary breast cancer. PCR analyses confirmed that the treatment led to significant reductions in human PLK1 mRNA in Her2+ tumors, but not Her2- tumors, without altering Her2 expression. Notably, treatment led to a marked reduction in the growth of Her2+ human cell line-derived tumors and primary breast cancer tumors, accompanied by reduced cell proliferation and increased apoptosis.
Similarly, in a separate set of trials in an experimental model of breast cancer metastasis, F5-P/PLK1 therapy reduced the development of lung and liver metastases, and increased survival. And while siRNAs can potentially activate nonspecific inflammatory responses by binding to innate immune RNA sensors, this wasn’t evident with the F5-P/PLK1 siRNA therapy either in vitro or in experimental mice. And in a final set of experiments, the researchers showed that using the carrier construct to deliver a cocktail of siRNAs targeting PLK1, CCND1, and AKT, demonstrated even greater tumor-blocking effects than PLK1 inhibition alone.
“Our findings provide preclinical support for the therapeutic potential of F5-P–delivered PLK1-siRNA for the treatment of Her2+ breast cancers,” Dr. Song et al conclude. “Because of the specificity of delivery, we were able to target a gene that is essential to dividing cells, without any obvious toxicity.”
The researchers suggest their method could also be adapted for the delivery of other siRNAs into cancer cells, either singly or as a mixture, providing new opportunities for personalized cancer therapy targeting an individual tumor type. Alternatively, the fusion protein’s tumor targeting antibody could be switched to target delivery of siRNAs to other cancer cell types.
Moreover, they point out, “The strong antitumor effects obtained here used a first-generation preparation that has not been optimized as to the choice of targeting antibody, siRNA, or dosing regimen. It is likely that optimization could lead to even better primary tumor and metastasis inhibition.”