As part of the ongoing quest to find effective delivery methods for RNAi drugs, a team of researchers at Duke University has combined aptamers with siRNAs to create a cell type-specific gene-silencing agent capable of inhibiting tumor growth and mediating tumor regression in an animal model of prostate cancer.
"The field has nicely shown that if you can get RNAi [molecules] into cells effectively, you can knock down gene expression for almost all targets," Bruce Sullenger, a Duke researcher and study co-author, told RNAi News this week. "The big issue from the therapeutic perspective is how to do that in vivo."
While numerous researchers are approaching the delivery problem from a variety of angles, Sullenger and his colleagues have taken an "all-RNA" tack in order to "make more of a universal … therapeutic."
"The basic idea was to take advantage of the RNA aptamers … which are really RNA ligands that bind to target proteins with high affinity and specificity … and link them to siRNAs," he said. "In a sense, [we are] using the aptamer as a localization signal, sort of a … guidance system for the siRNA, to get it into a particular cell type that is expressing a particular receptor on its surface."
In their paper, which appeared last week in Nature Biotechnology's online edition, the Duke researchers also note that additional benefits come from the fact that "aptamers and siRNAs have low immunogenicity … [that] can be easily synthesized in large quantities at a relatively low cost, and are amenable to a variety of chemical modifications that confer both resistance to degradation and improved pharmacokinetics in vivo."
|
"In a sense, [we are] using the aptamer as a localization signal, sort of a … guidance system for the siRNA, to get it into a particular cell type that is expressing a particular receptor on its surface." |
To test their approach, the researchers developed a molecule with the aptamer portion mediating binding to PSMA, a cell-surface receptor over-expressed in prostate cancer cells and tumor vascular endothelium, and the siRNA portion targeting the anti-apoptotic genes PLK1 and BCL2.
"We chose a receptor that was expressed on prostate cancer cells at high levels and is known to cycle to the inside of cells [at] high rates," Sullenger explained. "The idea was that you could use the aptamer/binding ligand portion to recognize the receptor on the surface of those prostate cancer cells … and deliver the siRNA cargo to the inside of the cells when the receptor was endocytosed."
According to the Nature Biotechnology paper, "depletion of the targeted gene products resulted in decreased proliferation and increased apoptosis of the targeted cells in culture … [while] cellular targeting of the chimeric RNAs was mediated by the interaction of the aptamer portion of the chimeras with PSMA on the cell surface.
"Notably, a mutant chimeric RNA bearing two point mutations within the region of the aptamer responsible for binding to PSMA resulted in loss of binding activity," the paper's authors added.
To try out the aptamer/siRNA approach in vivo, "we set up a human prostate cancer xenograft model in a nude mouse," Sullenger said. Direct injections of the therapeutic agent into the tumors induced tumor regression with no observed side effects.
"These reagents exhibited the same specificity for PSMA expression in vivo as they did in vitro, as the PSMA-negative PC-3 tumors did not regress when treated," the authors noted in the paper. "It is noteworthy that the RNA used to make the chimeras is protected from rapid nuclease degradation by the 2'-fluoro modification of the pyrimidines in the aptamer sense strand, which is likely to be essential for their performance."
Sullenger said that the next step for his team is to explore systemic delivery of the aptamer-siRNA chimeras
"There are certain cases where delivery directly into tumors would be useful, but the bigger utility would be systemic administration," he said. "The logical next step with the compounds we have would be to formulate them, synthesize them in large amounts, and do systemic delivery."
Additionally, the researchers are interesting in investigating how applicable the technology may be to other cancers, such as breast and blood cancers.
He said that although he and his colleagues are capable of conducting such research on their own, "there are a lot of oligonucleotide companies that have [put more focus] on the synthesis and formulation than we have," and a collaboration might make more sense.
"We've started to talk to a number of [parties] about establishing an interaction to explore that," including both RNAi drug developers and RNAi reagent shops, he said.
"Duke has filed provisional patent applications on the idea, so we're trying to work out the best approach to move the technology forward," Sullenger said. "Is it a collaboration with an RNAi company or is it to do it in house and just purchase reagents from one of the oligo synthesis places?"