Antisense Oligonucleotides Cross Rodents’ Blood-Mind Barrier

D.Delivering something therapeutic to the brain has long been a challenge, mainly due to the blood-brain barrier, a layer of cells that separates the vessels that supply blood to the brain from the brain itself. In a study published in Nature Biotechnology on August 12, researchers have now found that double-stranded RNA-DNA duplexes with attached cholesterol can enter the brains of mice and rats and change the concentration of target proteins. The results suggest a possible avenue for developing drugs that could target the genes involved in diseases such as muscular dystrophy and amyotrophic lateral sclerosis (ALS).

“It’s really exciting to have a study that focuses on central nervous system delivery” of systemically administered antisense oligonucleotides, says Michelle Hastings, who is studying genetic disorders at Chicago’s Rosalind Franklin University of Medicine and Science and isn’t participated in the study. . The authors “showed that it works for several goals, some of which are clinically relevant”.

See “Bringing Drugs Behind the Blood-Brain Barrier”

In 2015, Takanori Yokota from Tokyo Medical and Dental University and colleagues published a study that shows that a so-called heteroduplex oligonucleotide (HDO) – consisting of a short chain of DNA and an oligonucleotide with modified bases paired with complementary RNA that is linked to a Lipid is bound at one end – was successful in reducing target mRNA expression in the liver. Yokota’s team later worked with researchers from Ionis Pharmaceuticals to find out if HDOs could cross the blood-brain barrier and attack mRNA in the central nervous system.

In the new study, the research team showed that an HDO that targets a tumor-associated long non-coding RNA (Malate1) when labeled with cholesterol and intravenous injection had a higher knockdown score in the brain and spinal cord than when the HDO was labeled with the lipid tocopherol and subcutaneous injection. In both rats and mice, the effects of knockdown were related to the dose of HDO, and four doses one week apart were most effective. An intravenously injected single-stranded oligonucleotide bound to cholesterol could not destroy Malat1.

“Duplex oligos actually seem to deliver much better than single-stranded oligos,” said co-author Frank Rigo, Vice President of Functional Genomics and Drug Discovery at Ionis Pharmaceuticals, told The Scientist. “The data seem to suggest that the single-stranded oligos could be captured. . . and may not traverse the vasculature as efficiently as the double strand, ”he explains.

See “Oligonucleotide Therapeutics Shortly Before Approval”

After their initial results with Malat1, the researchers generated HDOs for three clinically relevant genes: DMPK, which, if mutated, is associated with a type of muscular dystrophy; glial fibrillar acidic protein (Gfap), which can cause Alexander disease; and human superoxide dismutase 1 (SOD1) which, when mutated and manipulated in mice, models amyotrophic lateral sclerosis. They found knockdowns of around 20 to 60 percent of the DMPK mRNA, depending on which central nervous system tissue they were analyzing. DMPK protein levels decreased by about half in both the brain and muscles of mice that received the HDO intravenously. The goals of the other two HDOs showed more modest knockdowns.

“The data appears to be strong,” wrote Rudy Juliano, an emeritus professor at the University of North Carolina School of Medicine who was not participating in the study, in an email to The Scientist. Open questions include whether the majority of the effects observed in the entire brain actually took place in neurons and not in supporting tissues, if the high doses used by the authors – 50 milligrams of HDO per kilogram of body weight – could be toxic, and why “this simple modification The oligostructure can overcome the extremely robust blood-brain barrier that restricts the access of much smaller and much less polar molecules to the brain. “

“The first thing I noticed was the enormous dose they were using. Fifty milligrams per kilogram is very high, ”agrees David Male, a cell biologist at the Open University in the UK who was not involved in the work. It is important to consider dosage for clinical uses, he adds. “You might want to combat inflammation in the brain, such as in multiple sclerosis. You will likely have to give fairly large doses over a long period of time if you are just giving an oligonucleotide that is not effectively self-regenerating. “

In the study, the authors recognize the limitations of current work, and Rigo says that optimization plans are already in place. “In theory, you want to find out how these lipids get into the brain. Which receptors and which systems do they use? ”He asks. “We want to expand the work and, if one or two receptors are used, find out which ones they are and then use them more specifically.”