The delivery of small interfering RNA (siRNA) cancer gene inhibitors via peptides may be improved through L/D-amino acid stereochemical modifications, according to Medical University of South Carolina (MUSC) Hollings Cancer Center researchers.

This work was done using the peptide 599, which previous studies showed was effective at delivering siRNAs to cancer cells to silence oncogenes. In this study, 599 was modified to generate eight different peptide variants, incorporating either different stereochemical patterns of L/D-amino acids or a specific D-amino acid substitution. These modifications could, in some instances, increase/decrease the binding, nuclease/serum stability, and complex release of siRNAs. They also increased the complex’s efficiency at gene-silencing.

This work was published online March 19 in Molecular Therapy – Nucleic Acids.

There are many technical challenges with siRNA delivery, said senior author Andrew Jakymiw, Ph.D., associate professor in MUSC’s Oral Health Sciences Department. “For example, rapid renal excretion, degradation by RNases, low intracellular uptake, endosomal entrapment and low release of the siRNA cargo from the delivery platform are all challenges that we must consider when modifying a peptide siRNA carrier.”

To harness siRNA’s gene silencing capabilities, scientists must get it into the appropriate cells, which requires attaching it to a larger molecule to protect it during delivery. Peptide carriers are an attractive tool for delivering siRNA, because they are affordable and easy to modify.

In earlier studies, the Jakymiw laboratory found that 599, a peptide carrier they designed, could deliver the siRNA cargo into cancer cells and turn off a targeted cancer gene, which inhibited tumor growth in a mouse cancer model.

“We originally designed the 599 peptide so that it could help the siRNA cargo penetrate the cell and escape endosomes more easily. However, by looking at the three-dimensional arrangement of the amino acids in the 599 peptide, in particular their stereochemistry, we were able to make additional changes that beneficially affected the peptide carrier’s capabilities,” said Jakymiw.

Charles Holjencin, a dual D.M.D./Ph.D. student in the Jakymiw lab, used confocal fluorescence microscopy and observed that one of the modified 599 siRNA-loaded peptide carriers, called RD3AD, was arranged around the cancer cells in a clear pattern that he had not seen with the original 599 peptide carrier.

The modified RD3AD peptide carrier was delivering the siRNA drug by adhering to and possibly moving along cell surface protrusions, called filopodia. Entry into the cell via filopodia is a very efficient way for small biological complexes to enter cells; some viruses and bacteria also use this entry method. RD3AD was able to enter cancer cells more efficiently and improved gene silencing.

One of the next steps will be to test the RD3AD peptide in animal cancer models. Additionally, the researchers want to understand the mechanisms associated with this form of drug delivery more fully. For example, an unanswered question is what protein is the peptide carrier interacting with on filopodia? If this molecule is overexpressed in cancer, then this could be a valuable therapeutic target, especially for aggressive cancers, which typically have increased numbers of filopodia.

While cancer cells were the biological target for improving this drug delivery system, peptide carriers, such as RD3AD, have more possible applications. In fact, they could be used to deliver siRNA in any instance where gene silencing is desired for the treatment of a disease.