Antisense Oligonucleotides Make Sense

Antisense oligonucleotides (ASOs) are short (about 12 to 25 nucleotides long), synthetic single strands of DNA or RNA that are complementary to a chosen sequence. They can alter RNA and reduce, restore, or modify protein expression. ASOs interact with proteins on the surface of cells and enter the cytoplasm. Then they can work either in right in the cytoplasm or enter the nucleus.

In their naked form, ASOs cannot permeate the plasma membrane and are highly sensitive to degradation by endonucleases and exonucleases. To overcome these problems, ASOs have been chemically modified. On the basis of these modifications, ASOs can be broadly classified into three generations.

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1. In first-generation ASOs, the phosphate backbone linking the nucleotides is modified. One of the non-bridging oxygen atoms in the phosphodiester bond is replaced by a sulfur, methyl or amine group. These chemical modifications have improved the ASO stability by increasing the resistance of ASOs to nucleases. Unfortunately, the biologically active modified ASOs are highly toxic due, in particular, to their non-specific binding to proteins. This led researchers to develop new generations of ASOs that were both less toxic and more specific.


2. The second generation of ASOs is characterized by alkyl modifications at the 2′ position of the ribose. These ASOs are less toxic and have a slightly higher affinity for their target.


3. The third generation is more heterogeneous because it includes a large number of modifications aiming to improve binding-affinity, resistance to nucleases, and pharmacokinetic profile. The most common modifications include locked nucleic acids; phosphorodiamidate morpholino oligomers, in which the ribose is replaced by a morpholine moiety and the phosphodiester bond by a phosphorodiamidate bond; and peptide nucleic acids, in which the ribose-phosphate backbone is replaced by a polyamide backbone.

Several distinct mechanisms of ASOs are known:

1. Activation of RNase H. It is the most commonly used mechanism of action of ASOs. Such ASOs work in the cytoplasm. Antisense RNAs prevent protein translation of messenger RNAs by binding (hybridizing) to them. After binding, the hybrid can be degraded by a specific enzyme, RNase H, which hydrolyzes RNA. Such approach results in 80-95% down-regulation of mRNA expression. Volanesorsen is an example of antisense RNAs. It is a triglyceride-reducing drug that targets the mRNA for a protein that slows down the breakdown of fats called apolipoprotein C3. By blocking the production of this protein, the medicine reduces the level of triglycerides in the blood and, as a result, fat accumulation in the body.


2. Alteration of splicing. After DNA is transcribed into the precursor form of mRNA (pre-mRNA), the pre-mRNA must be spliced to exclude non-coding introns and exclude or include specific exons, generating the mature mRNA. Alterations in the appropriate splicing pattern can lead to disease. ASOs can block the splicing machinery from inappropriately deleting certain sequences. ASOs of this type need to penetrate into the nucleus. Duchenne muscular dystrophy (DMD) is a disease caused by “wrong” splicing. Inappropriate deletions alter the translational reading frame of the protein dystrophin, which is required for the integrity of the sarcolemma membrane. Most DMD patients are unable to walk by the age of 12 and die before turning 30 due to respiratory and/or cardiac failure. Eteplirsen, the ASO approved for treatment of DMD, has the unique ability to repair the RNA by promoting splicing that leads to the correct RNA sequence, that is inclusion of the right exons. 


3. Inhibition of 5′ cap formation. These ASOs also work in the cytoplasm. An ASO is designed to target sequences in the 5’ UTR. It binds near the cap site of pre- mRNA, preventing proteins (for example, translation initiation factor eIF-4α) needed for cap formation from binding. Ribosome association with eIF4- α triggers recruitment of the translation machinery. Therefore, inhibition of eIF-4α binding prohibits 5’cap-dependent translation. Such ASOs are still tested in animal models.


4. Increasing translation. An ASO blocks upstream open reading frames or other inhibitory elements in the 5’UTR, increasing translation efficiency. There are no approved or close to being approved ASOs acting to boost translation.


5. Steric blocking of translation. These ASOs also work in the cytoplasm.  Steric blocking of mRNA translation is usually achieved by designing ASOs that bind at or near the initiation codon of the mRNA sequence and hinder the translation machinery (ribosomal subunits) from binding. They are second-generation ASOs. These ASOs block access to mRNA but do not activate degradation of the target mRNA. So far, it has been little used in the development of drugs.

Here is a list of conditions that are hoped to be treated/ameliorated via antisense therapy: hematologic malignancies, myotubular and centronuclear myopathies, hereditary spastic paraplegias, inflammatory bowel disease, DMD, spinal muscular atrophy, spinocerebellar ataxias, autosomal dominant optic atrophy, autosomal dominant polycystic kidney disease, tuberous sclerosis, Parkinson disease, Alzheimer disease, tauopathies, amyotrophic lateral sclerosis, Huntington disease, familial amyloid polyneuropathy, gliomas, Dravet syndrome, acromegaly, rheumatoid arthritis, myotonic dystrophy, neoplasms liver metastases, lung cancer, renal cell cancer, bladder cancer, urothelial carcinoma, colorectal cancer, breast cancer,  ovarian cancer, prostate cancer, skin melanoma, type 2 diabetes mellitus, β-thalassaemia, paroxysmal atrial fibrillation, myelodysplastic syndromes, asthma, cystic fibrosis, Crohn's disease and others.  

There are quite a many companies and institutions racing to break into the market of ASO drugs, including but not limited to: Ionis Pharmaceuticals Inc. (an established leader in antisense drug development; lately, it has acquired Akcea Therapeutics, another developer of ASO therapeutics); Secarna Pharmaceuticals (leading European antisense drug discovery company); Antisense Therapeutics (working at products in-licensed from Ionis Pharmaceuticals Inc.); NeuBase Therapeutics (developing not only ASOs per se, but also focused delivery of them to target tissues; in addition, it is the first and only company to successfully create bifacial ‘Janus’ bases, which are engineered bases targeting double-stranded DNA or RNA by engaging both strands at once); GlaxoSmithKline; Stoke Therapeutics (striving to apply the splicing switching technology to a broader spectrum of genetic diseases); Isarna Therapeutics (GmbH); Imperial College London; Thomas Jefferson University; Enzon Pharmaceuticals, Inc.; INSYS Therapeutics Inc.; National Cancer Institute (NCI); Bio-Path Holdings, Inc.; Queen Mary University of London; Sterna Biologicals GmbH & Co. KG; ProQR Therapeutics; Glen Research (specializing in reagents for the synthesis, purification, modification and labeling  of DNA and RNA oligonucleotides); Genta Incorporated; Abramson Cancer Center of the University of Pennsylvania; OncoGenex Technologies; University of Chicago; NCIC Clinical Trials Group; British Columbia Cancer Agency; European Organisation for Research and Treatment of Cancer – EORTC; the University of Texas; Health Science Center at San Antonio; Eastern Cooperative Oncology Group; Pharmaxis; Aptose Biosciences Inc.; MedImmune LLC; Eleos, Inc.; AstraZeneca; Aegera Therapeutics; Daiichi Sankyo Co., Ltd.; Bio-Path Holdings (an oncology-focused biotechnology company refining its innovative technology to deliver DNA therapeutics directly to cancer cells).

Among these companies, there are antisense therapy ‘sharks’, like Ionis Pharmaceuticals, which was founded in 1989 by Stanley T. Crooke, who is regarded as a pioneer in the field of RNA-targeted therapeutics, and new entrepots, like Secarna Pharmaceuticals, which was founded in 2015, has come a long way for these years and risen to eminence in the ASO drug development, creating a diversified pre-clinical pipeline. Successful activities of companies with longtime expertise and rapid advances of startups prove that ASO therapeutics is a rapidly developing and promising area that has a potential to accommodate different unmet needs of modern medicine.

Why should ASOs be preferred over conventional treatment? There are few (or no) therapies for most orphan diseases. Antisense approaches would be a great asset for such diseases, as shown by the development of therapy for neuronal ceroid lipofuscinosis 7 (a rare autosomal recessive disorder). Genetic testing of the patient revealed mis-splicing of the CLN7 gene). A 22-nucleotide ASO named Milasen was tested in patient fibroblasts to restore the altered splicing (patient-customized). This starkly demonstrated the efficiency of ASOs as individualized genomic medicine. ASOs are particularly interesting and appropriate tools in cancer treatment because of the versatility of their action and the possibility of generating target-specific ASOs. The latest progress in antisense therapy is encouraging. ASOs have the potential to revolutionize personalized medicine.