Growing evidence suggests that the development of various forms of dementia, including Alzheimer’s disease, may be linked to the build-up of a protein called tau within the brain. Yet the use of conventional small molecule approaches to tackle the development of these tangles of protein have so far achieved limited success. Just last year, promising anti-tau compound LMTX proved no better than a placebo at improving cognitive abilities of Alzheimer’s patients in Phase III clinical trials.
There is now renewed interest in alternative treatments based on short chains of nucleic acids known as antisense oligonucleotides, with recent findings suggesting that antisense treatment may not only halt the formation of new tau proteins, but reduce existing levels too.
Antisense oligonucleotides are synthetic strands of nucleic acids capable of binding to the messenger RNA (mRNA) of specific gene targets. By altering the normal function of mRNA, these oligonucleotide chains can potentially restore protein levels where natural expression is impaired and lessen the toxic effects of other proteins by reducing their expression or creating modified versions.
First demonstrated almost four decades ago, the concept of using short nucleic acids chains to modulate gene expression has long proved possible in the lab, but has taken some time to emerge as a viable treatment in the clinic.
In fact, it wasn’t until 1998 that the first gene-silencing antisense therapy was granted market approval by the U.S. Food and Drug Administration (FDA). The 21 oligonucleotide chain drug fomivirsen (Vitravene), developed by Isis Pharmaceuticals for the treatment of cytomegalovirus infections in patients with compromised immune systems, was seen as a significant breakthrough in antisense therapy, and fueled hopes that the product would be just the first of many to come.
But this initial excitement was followed by disappointment. For many years, no further antisense therapeutics were approved, and Vitravene was subsequently withdrawn as improvements in other retrovirals undermined demand for the treatment.
However, a recent flurry of market approvals suggest antisense oligonucleotides are now ready to make a real impact on human health. In 2013, the FDA approved anticholesterol drug mipomersen (Kynamro), and late last year, the FDA granted approval for eteplirsen (Exondys 51) for the treatment of Duchenne muscular dystrophy, closely followed by nusinersen (Spinraza), for the treatment of spinal muscular atrophy.
There is now growing interest in the use of antisense oligomers to treat various forms of dementia. Recent findings published in the journal Science Translational Medicine earlier this year demonstrated that, when administered to mice genetically engineered to produce a form of the tau protein similar to that found in Alzheimer’s patients, the antisense oligonucleotide Tau ASO-12 resulted in a reduction in tau levels. When injected at the base of the rodents’ spines, the drug successfully spread throughout the brain, significantly reducing the number of existing tau tangles and preventing the protein from spreading in older mice. Similarly positive results were observed in primates, with no apparent side effects encountered – encouraging evidence that may support future human trials.
These findings join a small number of preclinical studies into antisense treatment targeting the amyloid precursor protein associated with the build-up of amyloid plaques, another proposed mechanism for the development of Alzheimer’s disease.
Improving safety and efficacy through chemical modification
Early antisense oligonucleotides were predominantly based on synthetic unmodified DNA. Yet despite the success of these initial studies, the oligonucleotide chains were prone to targeting by endo- and exonucleases. For antisense therapies to be clinically effective, their resistance to degradation would have to be improved through chemical modification.
Some of the earliest modifications replaced phosphate (PO) linkages with phosphorothiate (PS) linkages, involving the substitution of one of the non-bridging oxygen atoms in the phosphate backbone with a sulfur atom. While such modifications improved their stability towards nucleases, a number of off-target cytotoxic effects were identified, potentially due to protein binding or complement activation.
Second generation modifications that alter the oligonucleotide ribose backbone have been developed to overcome these toxicity problems while maintaining stability and bioavailability. The most successful of these are considered to be 2’-O-methyl and 2’-O-methoxy-ethyl additions, which increase the affinity of the oligonucleotides to their target RNA, and offer more resistance to nuclease degradation, while reducing cytotoxicity.
Quality control using ion-pair reversed-phase chromatography
However, while chemical modifications have been essential in advancing the pharmacological potential of antisense oligonucleotides, they can make characterization challenging. PS substitutions, for example, introduce a chiral center at phosphorus, which in combination with the chiral centers in D-ribose produce diasteroisomeric pairs at each PS linkage. PO impurities resulting from unreacted oligonucleotides, and those formed from PS oligonucleotide oxidation are also common.
As the therapeutic applications of oligonucleotides broaden, the level of analytical characterization demanded by regulatory bodies is becoming increasingly stringent.
There are a wide variety of chromatographic column chemistries for oligonucleotides and amino acids analysis. One of the most commonly used analytical approaches for oligonucleotide characterization and quantitation is ion-pair reversed-phase (IPRP) chromatography. This technique utilizes an ionic interaction between the analyte and the ion-pair reagent to increase the hydrophobicity of the analyte. Because of its greater hydrophobicity, the analyte experiences a stronger interaction with the polar stationary phase, improving the retention of the analyte and resulting in enhanced separation.
IPRP chromatography offers high-resolution separation of charged analytes such as oligonucleotides and can be directly coupled to mass spectrometry for identification of target oligonucleotides and related impurities including failure sequences.
Improvements in column technology mean robust options are available that are based on hydrophobic, polymer resins. These resins are stable under high pH and high temperature conditions which often provide higher resolution for challenging oligonucleotide samples.
Advances in synthetic methods have resulted in significant improvement in the safety and efficacy of antisense oligonucleotide treatments, with a number of exciting antisense dementia therapies in the pipeline. With growing regulatory scrutiny over the characterization of antisense treatments, ion-pair reversed-phase chromatography offers the high-resolution analytical performance that may help to accelerate the development of these innovative therapeutics and make them a clinical reality.
Kyle D’Silva, Ph.D. is a former analytical chemist who specialized in researching human dietary exposure to environmental contaminants using advanced spectroscopic techniques for a United Kingdom government research and reference laboratory. D’Silva’s mass spectrometry instrumentation expertise has taken him on an exciting journey through several leading instrument vendors in applications, product management and product marketing roles. D’Silva is now responsible for marketing connected analytical workflow solutions for the Pharmaceutical and Biopharmaceutical markets. He holds a Bachelor of Science degree in Color Chemistry, a Master of Science degree in Analytical Chemistry and Instrumentation, and a Ph.D. in research involving applied analytical chemistry and environmental sciences.