Oligonucleotide Therapeutics


    One of the most overlooked drug development platforms being used today lies in oligonucleotide therapeutics. Unlike small molecules, biologics and medical devices, oligonucleotides should strictly be classed into a system all on their own. Oligonucleotides are short, chemically synthesized nucleic acids of between 10 and 30 units in length. They have been used in research for over half a century (including by myself), mainly for their ability to modify gene expression. It is this property that makes them appealing for treating diseases, especially ones that are genetically inherited.

    In 1968 the Nobel Prize for physiology was awarded to Robert Holley, Har Gobind Khorana and Marshall Niremberg for their contributions to the understanding of how the genetic code is used for protein synthesis. Khorana was one of the first scientists to synthesize a DNA duplex using oligonucleotides. In the 1970s and 1980s, discoveries were made into how oligonucleotides could bind to RNA to block translation and eliminate gene products.

    A variety of oligonucleotides have been created and they are classed into four main categories according to mechanisms of action.

    1. Antisense oligonucleotides (ASOs) are the most common. These are single stranded DNA or RNA molecules that target messenger RNA (mRNA) inside the nucleus. ASOs can speed up mRNA degradation by activating cytosol RNAase H (Gapamer), block mRNA translation before ribosomes can perform translation (antagomir and steric block) or they can alter splicing to restore stability or function of healthy proteins (splice switching).

    Antisense oligonucleotides are chemically modified deoxynucleotides, like those of DNA or rbonucleotides (RNA): Their sequence is 3' to 5' so they are complementary to the sense sequence of a molecule of mRNA, 5' to 3'. They can physically block ribosomes from moving along the mRNA, preventing synthesis of the protein, they can hasten the rate of mRNA degradation or they can prevent splicing errors.

    2. Small interfering RNAs (siRNAs) are double stranded RNAs that also target mRNA. They hijack the gene-silencing pathway in cells to trigger degradation of unwanted mRNA that harbor mutations.

    3. Micro RNAs (miRNAs) are small non-coding RNAs that regulate post transcriptional gene expression. They can block activity of harmful miRNAs (anti-MiRs) in diseased bodies or they can mimic healthy miRNAs that need to be unpregulated.

    4. Aptamers form 3D structures that bind to tertiary structures of proteins. They act as ligands to help for protein entry into cells and vesicles. They can be conjugated to oligonucleotides to aid entry into the cell.

    Types of oligonucleotide therapies:

    In the early days of oligonucleotide therapy development there were many hurdles that prevented progress towards marketing approval. Molecules were unstable and easily broken down by endonuclease enzymes, a naturally occurring enzyme in the bloodstream. Systemic delivery was therefore ineffective.

    Later on, new drug delivery systems were engineered. Oligonucleotides were packaged into liposomes and lipid nanoparticles, delivered directly to the liver. This overcame much of the bioavailability problem. ASOs have been conjugated to sugars such as N-Acetylgalactosamine (GalNAc), to increase potency. Conjugated siRNA was developed using dynamic polyconjugate technology (DPC), to link sugar chains to protect the RNA molecule. Adeno-associated viral vectors have also been used to package oligonucleotides to enhance targeted delivery.

    Strategies for improving oligonucleotide delivery:

    To date only four oligonucleotide therapies have been approved by the FDA and have remained on the market. These include:

    Pegaptanib (approved in 2004, for OSI Pharmaceuticals) - treats neovascular age-related macular degeneration. Pegaptanib is an Aptamer which binds to VEGF proteins blocking its activity. It is delivered through intravitreal injection into the eye.

    Mipomersen / Kynamro (approved in 2003, for Ionis and Genzyme) - treats hypercholesterolemia. Mipomersen is an RNAse H stimulator which mediates cleavage of the cholesterol inducing Apolipoprotein B mRNA. This is delivered through subcutaneous injection.

    Eteplirsen / Exondys 51 (approved in 2016, for Sarepta Therapeutics) - treats Duchenne muscular dystrophy. Etelplirsen induces exon skipping which induces the expression of truncated but functional dystrophin. It is delivered through intravenous infusion. Eteplirsen had a particularly controversial approval pathway at the FDA since the clinical trial involved just 12 patients and a number of safety and efficacy questions were raised. It was originally voted down by the FDA Advisory Panel before approval.

    Nusinersen / Spinraza (approved in 2016, for Ionis and Biogen) - treats Spinal muscular atrophy. Nusinersen mediates exon 7 inclusion and by doing this produces more functional SMN protein. It is delivered through intrathecal injection following a spinal tap.

    Oligonucleotide therapies past and present:

    One other ASO therapy was approved back in 1998 called Fomivirsen. This was used to treat cytomegalovirus retinitis in HIV patients. It acted by mediating cleavage of human CMV mRNA and had to be delivered by intravitreal eye injection. However this was discontinued after competition from more effective HIV drugs drove it out of the market. Pegaptanib also met a similar fate when better therapies were achieved through monoclonal antibodies.

    The recent approvals of Eteplirsen and Nusinersen mark a new chapter in the revival of oligonucleotide therapies. It is hoped that better delivery systems being developed will enhance bioavailability and potency of these molecules. One has only to look at clinicaltrials.gov to find that over 100 clinical trials are currently ongoing for over 70 oligonucleotide therapies. Market research predicts RNAi therapeutics to reach $4.58 billion by 2022.


    Catherine Offord, The Scientist, Dec. 2016; Oligonucleotide Therapeutics Near Approval

    Godfrey et al., EMBO Mol Med. 2017 Mar 13. pii: e201607199. Delivery is key: lessons learnt from developing splice-switching antisense therapies

    Lundin et al., Hum Gene Therapy. 2015, 26:8, 475-485. Oligonucleotide Therapies: The Past and the Present

    Corey DR., Nat Neurosci. 2017 Feb 13. doi: 10.1038/nn.4508. Nusinersen, an antisense oligonucleotide drug for spinal muscular atrophy

    Zhao W et al., Drug Des Devel Ther. 2016 Nov 24; 10:3851-3865. Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery.