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RNA Based Therapeutics

Over the past decades RNA based therapeutics gained importance. Especially in diseases with genetic causes, oligonucleotide chains offer a promising therapy approach - often the first ever. Currently there are two main approaches used to target RNA: double stranded RNA-mediated interference (RNAi) and antisense oligonucleotides (ASO). Both approaches are currently in clinical trials for targeting of RNAs involved in various diseases, such as cancer and neurodegenerative diseases.

Challenges in Therapeutic Production

Due to the sequence specificity, an oligo therapeutic can in principle be used against any mRNA and thus against any disease treatable by gene knockdown. However, the idea of oligos also comes with downsides. Problems are the comparatively rapid degradation of RNA in the bloodstream and the targeted uptake of the therapeutic, if possible, only in diseased cells or tissues of an organism.

The evolution of the medicinal chemistry of oligonucleotides has been critical to the steadily improving performance of ASOs in the clinic. ASOs are oligomeric and comprised of nucleotide analogs. Because ASOs can be designed to work through a variety of post-RNA-binding mechanisms, numerous designs have been evaluated. As new molecular mechanisms of action are identified and new insights into the molecular mechanisms of distribution, cellular uptake and subcellular distributions, and various toxicities are reported, the designs are becoming progressively more complex.

Case Study: Antisense Oligonucleotide Silencing of FUS

Fused in sarcoma (FUS) is an RNA-binding protein that is genetically and pathologically associated with rare and aggressive forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Korobeynikov et al researched on ION363, a non-allele-specific FUS antisense oligonucleotide. ION363 efficiently silences Fus and reduces postnatal levels of FUS protein in the brain and spinal cord, delaying motor neuron degeneration. In mouse genetic and human clinical studies, evidence is provided in support of FUS silencing as a therapeutic strategy in FUS-dependent ALS and FTD.

Oligonucleotide Medicinal Chemistry: Modifications and their Advantages

The PS modification is broadly used in all major classes of ASOs and all chemically modified siRNAs. The replacement of one nonbridging oxygen with a sulfur alters the physicochemical characteristics of the phosphate in important ways. Because the sulfur atom is twice as large as the oxygen atom, the charge distribution, bond angles, and stretching of PS links differ substantially from PO linkages. The sulfur substitution spreads the charge and makes the phosphate more “lipophilic,” thereby facilitating binding to proteins. As a general rule, for proteins that require PS moieties to bind ASOs, the minimum number of this modification needed to support meaningful protein interactions is 10. The enhanced protein binding enabled by PS substitutions is critical as protein binding of ss PS ASOs plays crucial roles in the absorption, distribution, cellular uptake, intracellular distribution, activity, and toxicity of PS ASOs.

Modifications at the 2′ position of the ribose ring are commonly used to help increase oligonucleotide stability and improve resistance to nuclease activity in vivo. RNA oligonucleotides synthesized using 2′-MOE modifications phosphoramidites have shown to be more nuclease resistant, with lower toxicity, and slightly increased hybridization affinities, making them well suited for therapeutic in vivo applications, such as ASO, siRNA, and aptamers. 2'-O-methylnucleotides offer advantages in kinetic and melting properties oligoribonucleotide probes. 2'-O-Methyl oligoribonucleotide probes bind to RNA targets faster and with much higher melting temperatures at various probe length. Because of their greatly enhanced Tm when bound to RNA, 2'-O-methyl oligoribonucleotide probes can efficiently bind to double-stranded regions of structured RNA molecules. The increased melt, faster kinetics of hybridization, ability to bind to structured targets and increased specificity of 2'-O-methyl oligoribonucleotide probes render them superior to corresponding 2'-deoxy oligoribonucleotides for use in assays that detect RNA targets.

PS: Phosphorothioate

 phosphorothioate backbone-modified oligonucleotides

2’-OMe: 2'-O-methylated Nucleosides

 phosphorothioate backbone-modified oligonucleotides

2’-MOE: 2-Methoxyethyl

 phosphorothioate backbone-modified oligonucleotides

2’-LNA: Locked Nucleic Acid

 phosphorothioate backbone-modified oligonucleotides

2′-MOE-modified oligonucleotides demonstrated that the oligonucleotides exhibited a tissue distribution similar to phosphorothioate oligodeoxynucleotides (PS ODNs) and decreased toxicities compared to PS ODNs. Additionally, 2ʹ-MOE substitution significantly reduces proinflammatory effects.

Therapeutic viability of siRNA gene silencing is dependent on improvements in molecule bio-stability, specificity and delivery. Modification of siRNA with Locked Nucleic Acid (LNA), a nucleic acid analogue with unprecedented binding affinity overcomes this downfalls. LNA provides excellent specificity toward complementary RNA and DNA oligonucleotides. Incorporation of LNA substantially enhances serum half-life of siRNA's, which is a key requirement for therapeutic use. Moreover LNA is compatible with the intracellular siRNA machinery and can be used to reduce undesired, sequence-related off-target effects. The remarkable properties of LNA have led to applications within various gene silencing strategies both in vitro and in vivo.

Reliable Detection of Modified Oligos

In production processes of therapeutic agents, rigorous and consitent quality control is key. antibodies-online, together with out partner Rockland Immunochemicals, is able to support your development quality control processes. Rockland’s ModDetect™ Panels are antibody reagent panels developed to detect oligo modifications independent of the sequence or location of the modification. This makes the ModDetect™ Panels useful for oligonucleotide therapeutic development, mRNA vaccine development, or research of genetic diseases or gene expression.

Get in touch with our experts at antibodies-online and Rockland to discuss antibodies against oligo modifications customized for your needs.

Discover our Services for anti-Oligo Modification Antibodies!

References

  1. Iwamoto, Butler, Svrzikapa, Mohapatra, Zlatev, Sah, Meena, Standley, Lu, Apponi, Frank-Kamenetsky, Zhang, Vargeese, Verdine: "Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides." in: Nature biotechnology, Vol. 35, Issue 9, pp. 845-851, (2017) (PubMed).
  2. Crooke, Wang, Vickers, Shen, Liang: "Cellular uptake and trafficking of antisense oligonucleotides." in: Nature biotechnology, Vol. 35, Issue 3, pp. 230-237, (2017) (PubMed).
  3. Derbis, Kul, Niewiadomska, Sekrecki, Piasecka, Taylor, Hukema, Stork, Sobczak: "Short antisense oligonucleotides alleviate the pleiotropic toxicity of RNA harboring expanded CGG repeats." in: Nature communications, Vol. 12, Issue 1, pp. 1265, (2021) (PubMed).
  4. Crooke, Liang, Baker, Crooke: "Antisense technology: A review." in: The Journal of biological chemistry, Vol. 296, pp. 100416, (2021) (PubMed).
  5. Crooke, Baker, Crooke, Liang: "Antisense technology: an overview and prospectus." in: Nature reviews. Drug discovery, Vol. 20, Issue 6, pp. 427-453, (2021) (PubMed).
  6. Majlessi, Nelson, Becker: "Advantages of 2'-O-methyl oligoribonucleotide probes for detecting RNA targets." in: Nucleic acids research, Vol. 26, Issue 9, pp. 2224-9, (1998) (PubMed).
  7. Elmén, Thonberg, Ljungberg, Frieden, Westergaard, Xu, Wahren, Liang, Ørum, Koch, Wahlestedt: "Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality." in: Nucleic acids research, Vol. 33, Issue 1, pp. 439-47, (2005) (PubMed).
  8. Chery: "RNA therapeutics: RNAi and antisense mechanisms and clinical applications." in: Postdoc journal : a journal of postdoctoral research and postdoctoral affairs, Vol. 4, Issue 7, pp. 35-50, (2016) (PubMed).
  9. Korobeynikov, Lyashchenko, Blanco-Redondo, Jafar-Nejad, Shneider: "Antisense oligonucleotide silencing of FUS expression as a therapeutic approach in amyotrophic lateral sclerosis." in: Nature medicine, Vol. 28, Issue 1, pp. 104-116, (2022) (PubMed).
  10. Dovgan, Koniev, Kolodych, Wagner: "Antibody-Oligonucleotide Conjugates as Therapeutic, Imaging, and Detection Agents." in: Bioconjugate chemistry, Vol. 30, Issue 10, pp. 2483-2501, (2020) (PubMed).
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