Researchers from the Novartis Institutes for Biomedical Research in Switzerland have developed a method called whole-body scanning PCR to better analyze the way candidate oligonucleotide therapeutics distribute themselves throughout the bodies of mice.
The team, led by first author Julien Boos, described the approach in a study published last week in Nucleic Acids Research. According to the researchers, the WBS-PCR platform is compatible with a variety of downstream PCR and other detection methods for both wild-type and chemically modified oligonucleotides, as well as endogenous RNA molecules.
In the study, the researchers demonstrated the approach with both standard RT-qPCR techniques and novel PCR methods they designed to work with specific types of candidate molecules. The researchers wrote that the method could also be expanded to work with aptamer-based detection systems to scan for distribution of peptides and proteins.
Iwan Beuvink and Juerg Hunziker, co-senior authors of the paper, told PCR Insider this week that the group was interested in developing a more opportunistic and comprehensive approach than current methods focusing on single organs or in vitro cell-based assays to identify the tissue targets of new oligonucleotide therapeutics.
Drug companies, including Novartis, are increasingly working with these molecules as potential therapies, but their development has been challenging — especially ensuring effective and specific delivery to various tissues.
"Novartis, of course, has activities in these directions, but the development of this method was more to give the global scientific community a tool," said Beuvink.
"A recurring theme in all these efforts is delivery: how you can deliver the siRNAs or other oligos into the tissues where you'd like to have them biologically active," Beuvink said.
"Several efforts have been undertaken to either use antibodies, or lipid formulations, or put homing peptides on them, and some of these seem to be working, but there was no real platform available that allowed you to screen large quantities of these molecules," he explained. "A lot of people are looking and not many people are finding."
The Novartis researchers aimed for method that would potentially allow them to inject a battery of therapeutic candidates into mice, then section the animals to see everywhere a signal may be appearing, indicating activity.
In the recently published study, the group focused on demonstrating the platform one molecule at a time using several types of oligos and PCR methods. However, the authors wrote that moving forward they plan to work to multiplex the WBS-PCR approach.
The WBS-PCR process begins with sealing a thin cross-section of an oligo-treated mouse's body to a small area of a pre-filled 1,536-well plate. By inverting and centrifuging the plate, the target is extracted from the mouse tissues directly, and according to its position in the cross-section. Then, according to the authors, everything is transferred to a 384-well plate for downstream analysis using a chosen PCR method.
The location of the analyte is visualized by converting qPCR or other signals to an image and overlaying them with a picture taken of the whole-body mouse cross-section.
"In our manuscript we first validated the system looking at mRNAs with known tissue-specific distribution patterns," Beuvink said, using standard TaqMan RT-qPCR procedures.
The group also developed a novel RT-qPCR method optimized to look at smaller molecules, including microRNAs and siRNAs.
The researchers looked at a tritium-labeled siRNA in order to compare WBS-PCR to a method using quantitative whole-body autoradiography. They found that while the QWBA approach identified locations containing intact oligos and tissues containing associated metabolites, WBS-PCR resulted in a distribution pattern specific to intact molecules only.
According to the authors, this could prove valuable in reducing false positive tissue hits caused by the accumulation of metabolites in secondary tissues.
In a final experiment, Beuvink said, the researchers "took things a step further," by developing a method they call chemical-ligation qPCR to allow them to track the distribution of heavily chemically modified oligos.
CL-qPCR is a two-step assay that uses qPCR to quantify the amount of product formed in a self-directed chemical ligation of two oligodeoxynucleotides templated by the fully complementary analyte, according to the authors.
"The field is exploding at the moment with miRNA inhibitors," Beuvink explained. "And they have the problem that they are becoming less and less like natural molecules, so conventional PCR methods don’t recognize them as substrate anymore."
"Enzymes only work so far, so if something is getting too heavily modified, the enzymes won't do their job anymore," Hunziker added.
Beuvink declined to detail Novartis' plans in terms of using the method for drug development moving forward, but he highlighted the flexibility of the platform for analyzing different types of molecules using different PCR strategies as a boon to the field as a whole.
In addition to working on multiplexing the WBS-PCR approach, an important next challenge will be to develop the method to work with aptamers, which are also being investigated as therapeutics, as well as homing molecules for the delivery of other oligonucleotides, Beuvink said.