Antisense molecules

Zamecnik P 1978

Antisense oligonucleotides are chemically modified and thus synthetic, short-chain single-stranded nucleic acids that contain the complementary (and thus "contrary to the original sense" = anti-sense opposite) genetic information of a gene. They bind firmly to the messenger RNA of the gene, which means that translation thus influences or even prevents the conversion of the mRNA into a protein in the ribosomes and thus the biosynthesis of proteins (Benimetskaya L et al. 1997; Bennett CF et al. 2017).

Antisense oligonucleotides are spontaneously taken up by cells in small amounts. By using certain carriers (e.g. certain carrier lipids), an improvement in their uptake into the cell (transfection) and a better intracellular distribution can be achieved. The therapeutic effect of antisense oligonucleotides (ASO) has now been well documented in a wide range of clinical pictures (Askari FK et al. 1996). Perhaps their most significant advantage over other therapeutics is that knowledge of the gene target sequence directly provides knowledge of possible complementary oligonucleotide therapeutics. Thus, the concept of a targeted modulation of disease-causing proteins with antisense oligonucleotides has shown positive results in various clinical studies in viral, inflammatory and oncological diseases. In the last 5 years, more than 100 therapeutic approaches based on antisense oligonucleotides have been tested in Phase I clinical studies, of which about 25% have reached Phase II / III.

A good example of a positive therapeutic effect of this substance group is the antisense agent milasen, which is tailored to the gene mutation in Batten disease, a rare genetic defect that leads to neurological deficits.

Furthermore, ^Exondys51® is an antisense oligonucleotide for the treatment of Duchenne muscular atrophy.

Nusinersen (^Spinraza®), a causal, disease-modifying therapy for the treatment of 5q-associated spinal muscular atrophy (SMA), has been available on the German market for the first time since July 2017. This gene modulator increases the production of the complete and functional SMN protein, binds to the SMN2 pre-mRNA and thus promotes the inclusion of exon 7 in the mRNA. This enables the production of functional SMN protein. The half-life in the cerebrospinal fluid is 135 to 177 days (Finkel RS et al. 2017).

Of particular note are fomivirsen and mipomersen, which have been approved for the treatment of cytomegalovirus retinitis and familial hypercholsterinemia respectively (Adams BD et al. 2017). In the approval studies, treatment with mipomersen (Kynamro®), an antisense oligonucleotide (blocks the messenger RNA for the synthesis of apolipoprotein B in the liver) as an add-on therapy to statins at the primary efficacy time point (PET), led to a statistically significant improvement in the treatment response, PET) led to a statistically significant reduction in LDL-C levels of 24.7 % in patients with homozygous familial hypercholesterolemia (HoFH) and 35.9 % in patients with severe heterozygous familial hypercholesterolemia (HeFH) compared to the respective baseline values, which can be considered clinically relevant. The FDA voted in favor of approving this antisense oligonucleotide in 2012, while the EMA voted against it in 2013.

Fomivirsen (Vitravene®) is a 21mer antisense DNA phosphorothioate oligonucleotide that has been used since 1996 as an antiviral agent for the treatment of cytomegalovirus (CMV) infections in immunodeficiency (e.g. AIDS). Fomivirsen has a complementary sequence to the mRNA, the major immediate-early (MIE) transcriptional unit of the human cytomegalovirus (CMV). Binding of the aRNA to the complementary mRNA blocks the translation of this viral mRNA (Anderson KP et al. 1996). No protein synthesis takes place. Due to the double-strand formation, the cell's own enzyme ribonuclease H becomes active and cleaves the mRNA in this region. The oligonucleotide is then released again and can bind to another mRNA.

The orally administered antisense oligonucleotide (Mongersen) has been used in several clinical studies in patients with Crohn's disease. Mongersen inhibits SMAD7, a molecule that blocks the release ofTGF-ß1 (Monteleone G et al. 2015). SMAD7 is overexpressed in Crohn's disease, among others. This overexpression leads to disturbances in the "TGF-beta" signaling pathway whose undisturbed function has an anti-inflammatory effect. Blocking the TGF-beta signaling path way by overexpressed SMAD7 (or by CYLD) suppresses the function of regulatory T cells and leads to inflammatory reactions, a mechanism that is considered to be pathogenetically important in Crohn's disease. Mongersen is expected to be approved.

Antisense oligonucleotides in tumor diseases: In tumor diseases, several proliferation-promoting proteins (oncogenes) usually contribute to growth at the same time. Inhibiting individual oncogenic proteins is therefore not very promising. However, certain protein kinases play a superordinate role in the regulation of cell proliferation and differentiation. In leukemia cell lines, for example, inhibition of protein kinase A1 (PKA1) with an antisense oligonucleotide leads to a shift in the balance in favor of the expression of protein kinase A2 (PKA2).

Inhibition of Bcl-2 protein: Oblimersen (Genasense®; also known as augmeroses or bcl-2 antisense oligodeoxynucleotide G3139) is a synthetic single-stranded phosphorothioate DNA oligonucleotide with 18 bases that was developed to down-regulate bcl-2 mRNA expression (Klasa et al. 2002). Oblimersen selectively hybridizes to the first 6 codons of the open reading frame encoding the Bcl-2 protein and ultimately prevents translation of the Bcl-2 protein. Bcl-2- is a key molecule that is overexpressed by many tumor cells. This shifts the balance between survival and apoptosis towards the survival of the tumor cells. Thus, in many tumor entities, neoplastic cells can survive by protecting themselves from programmed cell death (apoptosis). By completely blocking Bcl-2, oblimersen can enhance the effect of cytostatic therapy against a number of tumor entities, such as chronic lymphocytic leukemia, B-cell lymphoma, breast carcinoma, bronchial carcinoma and malignant melanoma (Ott PA et al. 2013; Herbst RS et al. 2004). In later studies, however, the efficacy of this therapeutic principle in metastatic malignant melanoma could not be proven (Bedikian AY et al. 2014).

Non-antisense-mediated effects" of oligonucleotides: In addition to the specific antisense-mediated inhibition of a target protein, further effects of antisense oligonucleotides can be demonstrated that occur independently of the specific antisense effect. These so-called "non-antisense-mediated effects" of oligonucleotides are based on their direct binding to proteins (Hartmann G et al. 1997). Such effects can induce antiviral, anti-adhesive or immunostimulatory effects, among others.

The non-antisense-mediated immunostimulation of oligonucleotides is of therapeutic interest. This effect is due to general differences in the base composition of bacterial DNA and vertebrate DNA. In bacterial DNA, the two-sequence cytidine base followed by a guanidine base (CpG dinucleotide) occurs frequently. In vertebrate DNA, this two-strand sequence is less common and is modified by the addition of a methyl group to the cytidine base (in contrast to non-methylated bacterial DNA). Vertebrates recognize this difference in the base composition of DNA via a mechanism that has not yet been clarified. In vertebrates, the recognition of bacterial DNA leads to the physiological activation of a non-specific immune response. The immunostimulatory property of bacterial DNA can be imitated by synthetic oligonucleotides. A phosphorothioate modification of these oligonucleotides further enhances their immunostimulatory effect. Monocytes, macrophages and dendritic cells are also directly activated. In these cells, there is an increased synthesis of the cytokines tumor necrosis factor-alpha and interleukin-12. However, bacterial DNA or oligonucleotides with CpG dinucleotides have no direct influence on T lymphocytes.

In inflammatory (including oncological and viral) processes, proteins are known to contribute significantly to the pathogenesis of the respective disease. In inflammatory diseases, the inhibition of the formation of pro-inflammatory cytokines and leukocyte/endothelial adhesion molecules leads to a reduction in undesired inflammatory reactions. In acute and chronic inflammatory diseases, the pro-inflammatory tumor necrosis factor-alpha (TNFa) plays a central mediator role. The synthesis of TNF-alpha can be specifically inhibited in cell cultures with antisense oligonucleotides.

The continuous improvement of innovative RNA modifications as well as the improvement of release mechanisms (e.g. as nanoparticles) will open up the development of future RNA-based therapeutics for a broader spectrum of chronic diseases (Adams BD et al. 2017).

1. Adams BD et al (2017) Targeting noncoding RNAs in disease. The Journal of clinical investigation 127: 761-771
2. Anderson KP et al (1996): Inhibition of human cytomegalovirus immediate-early gene expression by an antisense oligonucleotide complementary to immediate-early RNA. Antimicrob agents Chemother 40: 2004-2011.
3. Askari FK et al (1996) Antisense oligonucleotide therapy. New Engl J Med 334: 316-320.Bedikian AY et al. (2014) Dacarbazine with or without oblimersen (a Bcl-2 antisense oligonucleotides) in chemotherapy-naive patients with advanced melanoma and low-normal serum lactate dehydrogenase: 'The AGENDA trial'. Melanoma research 24:237-243.
4. Benimetskaya L et al (1997) Mac-1 (cdllb/cd18) is a protein binding to oligodeoxynucleotides. Nat Med 1997; 3: 414-420.
5. Bennett CF et al (2017) Pharmacology of Antisense Drugs. Annual review of pharmacology and toxicology 57: 81-105.
6. Bennet CF (2019) Therapeutic Antisense Oligonucleotides are coming of age. Annu Rev Med 70:307-321
7. Finkel RS et al (2017) Treatment of infantile-onset spinal muscular atrophy with nusinersen: a phase 2, open-label, dose-escalation study. Lancet 388: 3017-3026
8. Hartmann G et al (1997) Playground of oligonucleotides: extrapolation from in vitro to in vivo? Nat Med 1997 (letter); 3: 702
9. Herbst RS et al (2004) Oblimersen sodium (Genasense bcl-2 antisense oligonucleotides): a rational therapeutic to enhance apoptosis in therapy of lung cancer. Clinical cancer research: an official journal of the American Association for Cancer Research 10: 4245-4248.
10. Li D et al (2018) Precision Medicine through Antisense Oligonucleotide-Mediated Exon Skipping. Trends in pharmacological sciences 39: 982-994.
11. Mercuri E et al (2018) Nusinersen versus sham control in later-onset spinal muscular atrophy. N Engl J Med 378:625-635
12. Monteleone G et al (2015) Mongersen, on Oral SMAD7 Antisense Oligonucleotides, and Crohn's Disease. N Engl J Med 372:1104-1113
13. Ott PA et al (2013) Oblimers in combination with temozolomide and albumin-bound paclitaxel in patients with advanced melanoma: a phase I trial. Cancer chemotherapy and pharmacology 71:183-191