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Despite Limitations, Gymnotic Delivery of Antisense Oligos Offers Benefits over Transfection

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By Doug Macron

Although transfection reagents are commonly used to deliver gene-silencing oligonucleotides to cells in culture, a relatively new technique that does not require delivery vehicles has been gaining traction.

Called gymnosis — from the Greek word "gymnos" for "naked" — the technique combines specifically modified antisense oligos with a cell's normal growth properties to achieve sequence-specific target knockdown. Though it is not without its limitations, gymnosis offers advantages over other approaches including RNAi in some situations, Albert Einstein-Montefiore Cancer Center's Cy Stein told Gene Silencing News.

Though antisense has been around for decades, it wasn't until a few years ago that researchers discovered gymnotic delivery. This was due in part because the necessary chemistries hadn't been invented, but also because the approach can take longer than many would expect, Stein explained.

In a Nucleic Acids Research paper published in 2007, Stein and colleagues from Santaris Pharma reported that locked nucleic acids — essentially nucleic acid analogs in which the ribose ring is locked by a methylene bridge connecting the 2'-O atom with the 4'-O atom — could silence target genes in even difficult-to-transfect cells in vitro with high efficiency and low toxicity.

Importantly, patterns of gene silencing triggered by gymnotically delivered oligos in vitro correlated better with in vivo silencing than those delivered using lipid transfection reagents.

According to Stein, gymnotic delivery appears to only work with phosphorothioate-modified, single-stranded oligodeoxynucleotides. The modification, he said, not only provides necessary nuclease resistance, but a “stickiness” that allows the molecules to adhere to the cell membrane.

But blocking nuclease sensitivity is only part of the equation. For reasons that aren't clear, the oligos must be further modified, either as LNAs or with a 2′-fluoro-beta-D-arabino nucleotide, or FANA, modification, he said. After that, the molecules can trigger gene silencing in a diversity of cells in culture without any transfection reagent or serum additives.

Gymnosis is far from a perfect approach, Stein noted. Perhaps the biggest limitation is that it can take days before an effect is seen in vitro, depending on the cell line being used — a curious effect considering that when the same oligo is tested in vivo, target knockdown occurs fairly rapidly in the same cell type.

Additionally, the amount of oligo required for gymnotic delivery is significantly greater than when using transfection reagents, which is not trivial considering the expense of LNAs, he said.

Nonetheless, gymnosis offers the benefit of a “cleaner readout” compared with lipid transfection, Stein said.

“Lipids have a way of [getting] into mitochondrial membranes, and when they do that, they cause toxicity,” he said. “But gymnotic delivery causes virtually no toxicity because the amount of material that's getting into cells in a topological sense is very low.”

Consequently, the in vitro activity of gymnotically delivered oligos correlates much better with in vivo activity than those that incorporate delivery agents.

“When screening a series of lipid-delivered oligonucleotides for activity against a particular target, “you overkill the target to such an extent that you get … a lot of false positives,” Stein said. “But with gymnotic delivery, you don't overkill the target ... so the oligos that work in vitro are really the ones that are the most active” in vivo.

Because of this, Stein said that he's heard that many of the big pharmaceutical companies have adopted the approach.

“I've been told that the big companies are all using gymnosis … [because] the cleanliness of the experiment beats transfection by lipid delivery vehicles,” he said.

“With lipids … you literally get thousands of gene changes,” regardless of the lipid used, he added. “If you use gymnotic delivery, you can cut those by 50 percent to 90 percent … [depending] on the system you're working in … and your gene target.”

Art Krieg, the former CSO of Pfizer's now-shuttered oligonucleotide therapeutics unit, confirmed that, at least at his previous company, this was the case.

“We found it useful for screening oligos in vitro,” he told Gene Silencing News. “There isn't a whole lot of work on [this] yet, but in our hands, when we were comparing different oligos in vitro and in vivo, the gymnotic delivery did seem to correlate better with in vivo activity than using transfection.”

But Krieg cautioned that the technique isn't perfect.

“It takes so long, you really have to be patient,” he said. And, again, less oligo is required with the use of transfection reagents.

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Still, gymnotic delivery is promising, and some of its limitations might ultimately be overcome with additional research, Krieg indicated.

“There is so little understood about the mechanisms of oligo uptake into cells, we just have a lot to learn there still,” he said.

Stein said that he is continuing his investigations into gymnosis, and it already appears that there may be ways to enhance the effect.

“Usually, if you do [something to get a cell to get it to] pick up oligonucleotides by gymnosis, the whole process just stops,” he said. “But we found some things that you can do to actually increase the efficiency, and that's telling us that there are … materials within the cell that contribute to this whole process that may be stimulated.”

It is also possible that with additional study, RNAi might benefit from gymnotic delivery.

As it stands now, gymnosis simply doesn't work with siRNAs for reasons unknown, although Krieg speculated that it might be because of differences in how RNA is taken up in cells versus DNA.

To Stein, it may be a problem of stickiness. “RNA molecules just aren't that sticky, and so they don't absorb through the cell surface that well,” he said. “As a result, what you'd be relying on with gymnotic delivery is essentially fluid phase endocytosis to get these things in, and it's not efficient.

“We tried some strategies to increase the phosphorothioate content of siRNAs, but it's never panned out,” he added. “The truth is, we haven't had the amount of time or resources to work on gymnotic delivery like people have worked on siRNAs, so there are all sorts of things we don't know.”

A recent Nucleic Acids Research paper by investigators at Isis Pharmaceuticals, however, might offer some clues as to the mechanism behind gymnosis.

According to that publication, the researchers were able to isolate a cell line called MHT from the livers of transgenic mice that maintained the same level of functional oligo uptake as that observed in vivo, and found that the uptake process and subsequent target knockdown appears to be dependent on the adaptor protein AP2M1.

“We have tested other cells like mouse embryo fibroblasts and H2.35 liver cells where inhibition of AP2M1 inhibited [single-stranded oligonucleotide]-mediated target reduction,” they wrote. “This shows that AP2M1 plays a similar role in cells other than MHT cells.”


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