The past influences the present……. and the future holds great promise

The ability to decrease (or knockdown) a disease-causing protein at its source vs. blocking a protein that is actively causing disease has fueled enthusiasm for antisense oligonucleotides, also known as ASOs. Knockdown remains one avenue; however equally exciting alternatives are now in sight. The approval of Spinraza® has shown an ASO can be life-saving for a severe disease in infants/children that affects muscle strength and movement. In this case the ASO directs a related, but non-functional, protein to become active and replace a damaged protein. ASOs are also in clinical development for ALS (or Lou Gehrig’s disease), Parkinson’s, Alzheimer’s, and many other diseases that directly impact quality and length of life. As expected, prior to starting clinical studies each ASO requires a robust nonclinical program; among toxicologists this raises questions such as “what is the best approach for nonclinical development to ensure the drug has adequate safety?”

ASOs take advantage of the specificity of Watson-Crick base pairing with a messenger RNA (mRNA) representing the sense strand and the ASO a short string of nucleotides (some modified) that are complimentary, or antisense. Manufacturing involves repeated chemistry steps to build a final length of 19 to 30 nucleotides. In this light ASOs are synthetic, as are small molecules. However, once in a cell an ASO interacts (hybridizes) with its target mRNA and impacts cell processes at specific steps. This aligns ASOs more with large molecules, also termed biologics. The combination of a synthetic manufacturing process and biologic-like actions means nonclinical development draws from both the similarities and differences between small and large molecules.

With small molecules a two species approach to identify possible risks to patients is expected. In fact, this has been the path for the majority of ASO programs. More in line with large molecules are concerns that loss of a protein or a change in protein function will result in adverse effects, often described as exaggerated pharmacology. The ideal study scenario is when at least one species can provide information on the risks associated with exaggerated pharmacology, as well as the general toxicity that may be present. In some programs this is not possible as the mRNA/protein is present only in humans, only when disease is active, or is unique to an individual patient. If the target is present, but the ASO is not active, a surrogate ASO designed for a specific species may be needed. These questions must be addressed on a case-by-case basis to achieve the proper balance between feasibility and a thorough evaluation.

Vetting ASOs for Human Testing

A typical IND-enabling program involves Maximum Tolerated Dose/Dose Range Finding studies, also called pilot toxicology, and GLP‑compliant definitive toxicology studies (such as a One Month Repeat Dose study). Safety pharmacology can be addressed within the toxicology studies or conducted as stand-alone studies. Genotoxicity includes standard in vitro and in vivo evaluations. Based on indication and patient populations, additional studies may also be needed to initiate Phase I studies, e.g., studies in juvenile animals. An analytical assay for dose solution concentration should be included. A bioanalytical assay for ASO concentration in blood allows for determination of exposure in the toxicology studies, and to make comparisons to potential exposure in humans.

One point that can be unique to an ASO is that time in blood can be relatively brief, hours to a few days, whereas the effects in cells can be long-lasting, days to weeks or even months. This is one reason tissue analysis is often included in ASO programs. Example tissues/organs include liver, kidney, spleen, lungs and heart as these tissues are known to accumulate ASOs or are often noted with histologic observations (e.g., basophilic granules, macrophage vacuolation, etc.) in toxicology studies. When possible, determination of pharmacology, such as knockdown of the target mRNA or intended change in the protein, are also included. The concentration of the ASO can be used to correlate magnitude and duration of exposure with magnitude and duration of pharmacologic and toxicologic effects. This data can provide further information on the potential effects in patients.

The above describes nonclinical programs that are representative of a typical ASO, but this drug class has undergone many changes and now includes non-typical approaches or involves new platforms such as short interfering (si)RNA, microRNA mimics, and microRNA antagonists. While having a close association with more traditional ASOs, the scope and focus of the nonclinical programs have adapted to cover the various indications, novel mechanisms of action, and delivery methods. The next blog in this ASO series will cover recent advances for these oligonucleotides.

Stay tuned!

If you are interested in learning more about the ASO pipeline, check out our recent Eureka blog article