Oligonucleotides (oligos) have been under clinical development for almost the past 30 years, beginning with antisense oligonucleotides (ASOs) and aptamers; and, followed about 15 years ago by silencing RNAs (siRNAs). Oligonucleotides, as peptides, resemble biologics in some ways, because of their molecular complexity, but are much smaller in size; leading to unique concerns in the design of control strategies for these types of molecules.
A good starting point to designing therapeutics in this class, is the usage of natural peptides to improve physical and chemical properties such as stability and bioavailability; and, in relation to methods of manufacture, chemical synthesis, recombinant DNA technology, or extraction from natural sources, continue to be the preferred methodology currently in use.
This is an extract from the whitepaper ‘CMC approaches to accelerate peptide, oligonucleotides and mRNA therapeutics development’ - download the whitepaper here.
The primary challenges
Despite considerable progress, two major obstacles stand in the way of widespread application of oligonucleotide therapeutics and regulatory approval: (i) drug safety, and (ii) delivery. The administration of oligonucleotides has been associated with the activation of innate immunity through interactions with toll-like receptors (TLRs); with some oligonucleotides binding to TLRs, and inducing immune responses similar to those induced by viral and bacterial RNA and DNA. Different sequence motifs have been identified as agonists of TLR family members; as such, avoiding these sequence motifs and using chemical modifications can minimize these immune-stimulatory effects.
The use of some siRNA therapeutics in clinical trials may be associated with another liability: inflammatory responses to the lipid nanoparticle formulations used to promote the uptake of siRNAs. Lipid nanoparticles are known to induce a complex antiviral-like response of innate immunity. To diminish the immune-stimulatory effects of the formulations, siRNAs in lipid nanoparticles have been administered in combination with antihistamines, non-steroidal anti-inflammatory drugs, and glucocorticoids.
A well-defined means of delivery is to directly conjugate a bioactive ligand to the RNA that will allow it to enter the cell of interest; and, perhaps the most clinically advanced example of this technique, is the conjugation of N-acetylgalactosamine (GalNAc), which targets the asialoglycoprotein receptor on hepatocytes, to siRNA.
This receptor-mediated uptake allows for lower dosing, than that required for the therapeutic delivery of unconjugated oligonucleotides, which is an essential feature for regulators approval and certification. Delivery to other cell types, such as muscle cells, can be accomplished by targeting antibodies or antibody fragments against cell-surface proteins known to be involved in intracellular transport.
Read the full whitepaper ‘CMC approaches to accelerate peptide, oligonucleotides and mRNA therapeutics development’ here.
- Stein CA and Castanotto D. FDA-Approved Oligonucleotide Therapies in 2017. Mol Ther. 2017;25:1069-1075.
- Cauchon NS, Oghamian S, Hassanpour S and Abernathy M. Innovation in Chemistry, Manufacturing, and Controls-A Regulatory Perspective From Industry. J Pharm Sci. 2019.
- Levin AA. Treating Disease at the RNA Level with Oligonucleotides. N Engl J Med. 2019;380:57-70.
- Behlke MA. Chemical modification of siRNAs for in vivo use. Oligonucleotides. 2008;18:305-19.
- Pepini T, Pulichino AM, Carsillo T, Carlson AL, Sari-Sarraf F, Ramsauer K, Debasitis JC, Maruggi G, Otten GR, Geall AJ, Yu D, Ulmer JB and Iavarone C. Induction of an IFN-Mediated Antiviral Response by a Self-Amplifying RNA Vaccine: Implications for Vaccine Design. J Immunol. 2017;198:4012-4024.
- Kaczmarek JC, Kowalski PS and Anderson DG. Advances in the delivery of RNA therapeutics: from concept to clinical reality. Genome Med. 2017;9:60.
- Nair JK, Willoughby JL, Chan A, Charisse K, Alam MR, Wang Q, Hoekstra M, Kandasamy P, Kel'in AV, Milstein S, Taneja N, O'Shea J, Shaikh S, Zhang L, van der Sluis RJ, Jung ME, Akinc A, Hutabarat R, Kuchimanchi S, Fitzgerald K, Zimmermann T, van Berkel TJ, Maier MA, Rajeev KG and Manoharan M. Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. J Am Chem Soc. 2014;136:16958-61.
- EMA. EMA Guidelines Relevant for Advanced Therapy Medicinal Products. 2019;2019.
- Sugo T, Terada M, Oikawa T, Miyata K, Nishimura S, Kenjo E, Ogasawara-Shimizu M, Makita Y, Imaichi S, Murata S, Otake K, Kikuchi K, Teratani M, Masuda Y, Kamei T, Takagahara S, Ikeda S, Ohtaki T and Matsumoto H. Development of antibody-siRNA conjugate targeted to cardiac and skeletal muscles. J Control Release. 2016;237:1-13.