Anti-sense oligonucleotides (ASOs) are short, modified, single-stranded oligonucleotides—made from DNA or locked nucleic acid (LNA)—with enhanced stability, activity, and bioavailability. They associate with RNA through sequence complementarity and can modify the expression (and/or splicing patterns) of target RNAs. These promising therapeutics are currently used to treat rare genetic diseases. Such RNA-based therapies are already being used successfully to treat previously incurable genetic disorders such as amyotrophic lateral sclerosis (ALS) and Duchenne muscular dystrophy.
A key challenge, however, is that most ASOs fail to reach their intended target within the cell. To target RNA, ASOs must cross membranes. Now, a new study reveals that slowing down the intracellular transport of RNA-based drugs may enhance their effectiveness.
This work is published in Nature Communications in the paper, “A CRISPR/Cas9 screen reveals proteins at the endosome-Golgi interface that modulate cellular anti-sense oligonucleotide activity.”
Using a genome-wide CRISPR/Cas9 knockout screen, researchers systematically knocked out thousands of genes to investigate their impact on ASO efficacy. “We identified a large number of genes that either improve or impair ASO activity,” said Liza Malong, PhD, senior scientist at Roche. “Many of these genes are involved in the intracellular transport of ASOs.”
Once administered, most ASOs are taken up by the cell and reach the cell’s endosomes via small transport vesicles. To exert their therapeutic effect, they must escape from the endosomes. Otherwise, they are declared as “cellular waste” and quickly shuttled to lysosomes for degradation. Since only a small fraction of ASOs manage to escape, their overall efficacy is limited. The likelihood of ASOs escaping from the endosomes is closely linked to the speed of intracellular transport: the longer they remain in the endosome, the more time they have to escape.
“By selectively switching off this gene, ASOs remain longer in specific endosomes,” explained Filip Roudnicky, PhD, a principal investigator at Roche. “This prolonged residence time increases their chance of escaping from the endosomes and becoming effective.”
The team discovered that the gene AP1M1 plays a key role in this process: it regulates the transport from the endosome to the lysosome. More specifically, the authors write that distinct targets, including AP1M1 and TBC1D23, link ASO activity to the transport of cargo between the Golgi and endosomes. “AP1M1 absence strongly increases ASO activity by delaying endosome-to-lysosome transport in vitro and in vivo. Prolonged ASO residence time in the endosomal system may increase the likelihood of ASO escape,” they write.
In both cell cultures and a mouse model, this approach significantly improved ASO efficacy without requiring an increased dosage.
“The key to more effective therapies thus lies not only in the drug itself, but also in intracellular trafficking,” added Anne Spang, PhD, professor at the Biozentrum at the University of Basel. “This concept may also apply to other drugs and even to bacterial and viral pathogens. Shortening the residence time of pathogens in endosomes could reduce their chance of escaping and replicating within the cell. This might represent a novel strategy in the fight against infections.”
The post Enhancing ASOs Efficacy by Slowing Down Intracellular Transport appeared first on GEN - Genetic Engineering and Biotechnology News.
A key challenge, however, is that most ASOs fail to reach their intended target within the cell. To target RNA, ASOs must cross membranes. Now, a new study reveals that slowing down the intracellular transport of RNA-based drugs may enhance their effectiveness.
This work is published in Nature Communications in the paper, “A CRISPR/Cas9 screen reveals proteins at the endosome-Golgi interface that modulate cellular anti-sense oligonucleotide activity.”
Using a genome-wide CRISPR/Cas9 knockout screen, researchers systematically knocked out thousands of genes to investigate their impact on ASO efficacy. “We identified a large number of genes that either improve or impair ASO activity,” said Liza Malong, PhD, senior scientist at Roche. “Many of these genes are involved in the intracellular transport of ASOs.”
Once administered, most ASOs are taken up by the cell and reach the cell’s endosomes via small transport vesicles. To exert their therapeutic effect, they must escape from the endosomes. Otherwise, they are declared as “cellular waste” and quickly shuttled to lysosomes for degradation. Since only a small fraction of ASOs manage to escape, their overall efficacy is limited. The likelihood of ASOs escaping from the endosomes is closely linked to the speed of intracellular transport: the longer they remain in the endosome, the more time they have to escape.
“By selectively switching off this gene, ASOs remain longer in specific endosomes,” explained Filip Roudnicky, PhD, a principal investigator at Roche. “This prolonged residence time increases their chance of escaping from the endosomes and becoming effective.”
The team discovered that the gene AP1M1 plays a key role in this process: it regulates the transport from the endosome to the lysosome. More specifically, the authors write that distinct targets, including AP1M1 and TBC1D23, link ASO activity to the transport of cargo between the Golgi and endosomes. “AP1M1 absence strongly increases ASO activity by delaying endosome-to-lysosome transport in vitro and in vivo. Prolonged ASO residence time in the endosomal system may increase the likelihood of ASO escape,” they write.
In both cell cultures and a mouse model, this approach significantly improved ASO efficacy without requiring an increased dosage.
“The key to more effective therapies thus lies not only in the drug itself, but also in intracellular trafficking,” added Anne Spang, PhD, professor at the Biozentrum at the University of Basel. “This concept may also apply to other drugs and even to bacterial and viral pathogens. Shortening the residence time of pathogens in endosomes could reduce their chance of escaping and replicating within the cell. This might represent a novel strategy in the fight against infections.”
The post Enhancing ASOs Efficacy by Slowing Down Intracellular Transport appeared first on GEN - Genetic Engineering and Biotechnology News.