Cancer is one of the leading causes of death worldwide, and conventional cancer therapies such as surgery, chemotherapy, and radiotherapy do not address the underlying molecular pathologies, leading to inadequate treatment and tumor recurrence. Angiogenic factors, such as EGF, PDGF, bFGF, TGF-β, TGF-α, VEGF, endoglin, and angiopoietins, play important roles in regulating tumor development and metastasis, and they serve as potential targets for developing cancer therapeutics. Nucleic acid-based therapeutic strategies have received significant attention in the last two decades, and antisense oligonucleotide-mediated intervention is a prominent therapeutic approach for targeted manipulation of gene expression. Clinical benefits of antisense oligonucleotides have been recognized by the U.S. Food and Drug Administration, with full or conditional approval of Vitravene, Kynamro, Exondys51, and Spinraza. Herein we review the scope of antisense oligonucleotides that target angiogenic factors toward tackling solid cancers.

Antisense oligonucleotide (AO) mediated exon skipping has been widely explored as a therapeutic strategy for several diseases, in particular, for rare genetic disorders such as Duchenne muscular dystrophy (DMD). To date, the potential of anhydrohexitol nucleic acid (HNA), cyclohexenyl nucleic acid (CeNA) and altritol nucleic acid (ANA) has not been explored in exon skipping. For the first time, in this study we designed and synthesised HNA, CeNA and ANA-modified 2′-O-methyl (2′-OMe) mixmer AOs on a phosphorothioate (PS) backbone, and evaluated their potential to induce exon 23 skipping in mdx mouse myotubes, as a model system. Our results clearly showed that all three AO candidates modified with HNA, CeNA and ANA could efficiently induce Dmd exon 23 skipping in vitro in parallel to the fully modified 2′-OMePS AO with reduced dual exon 22/23 skipping. In addition, they showed high nuclease resistance and no cytotoxicity compared to the 2′-OMePS AO, demonstrating the applicability of HNA, CeNA and ANA nucleotide-modified AOs in exon skipping.

Locked nucleic acid is a prominent nucleic acid analog with unprecedented target binding affinity to cDNA and RNA oligonucleotides and shows remarkable stability against nuclease degradation. Incorporation of locked nucleic acid nucleotides into an antisense oligonucleotide (AO) sequence can reduce the length required without compromising the efficacy. In this study, we synthesized a series of systematically truncated locked nucleic acid-modified 2'-O-methyl AOs on a phosphorothioate (PS) backbone that were designed to induce skipping exon 23 from the dystrophin transcript in H-2Kb-tsA58 mdx mouse myotubes in vitro. The results clearly demonstrated that shorter AOs (16- to 14-mer) containing locked nucleic acid nucleotides efficiently induced dystrophin exon 23 skipping compared with the corresponding 2'-O-methyl AOs. Our remarkable findings contribute significantly to the existing knowledge about the designing of short LNA-modified oligonucleotides for exon-skipping applications, which will help reduce the cost of exon-skipping AOs and potential toxicities, particularly the 2'-OMe-based oligos, by further reducing the length of AOs.

Serinol nucleic acid (SNA) is a novel nucleic acid analogue that can form highly stable heteroduplexes with complementary DNA and RNA sequences. Structurally, SNA is a close mimic to peptide nucleic acid (PNA) which is widely used in diagnostic and therapeutic applications. SNA chemistry is relatively new, and so far the scope of SNA has only been explored in improving the efficacy of small interfering RNA and for developing a highly sensitive molecular beacon for diagnostic applications. In this study, we investigated the potential of SNA-modified antisense oligonucleotide (AO) in parallel to PNA-oligo for splice-modulation in an in vitro cellular model of Duchenne muscular dystrophy (DMD). We synthesized a 20-mer SNA and PNA antisense oligonucleotide (AO) designed to induce exon-23 skipping in the mouse dystrophin gene transcript. Our results demonstrated that the SNA AO induced exon-23 skipping at all tested concentrations, whereas the corresponding PNA AO failed to induce any exon-23 skipping upon 24 hours of transfection using Lipofectin transfection reagent. Our results further expands the potential of SNA oligonucleotides in therapeutic applications.

Antisense oligonucleotide (AO)-mediated splice modulation has been established as a therapeutic approach for tackling genetic diseases. Recently, Exondys51, a drug that aims to correct splicing defects in the dystrophin gene was approved by the US Food and Drug Administration (FDA) for the treatment of Duchenne muscular dystrophy (DMD). However, Exondys51 has relied on phosphorodiamidate morpholino oligomer (PMO) chemistry which poses challenges in the cost of production and compatibility with conventional oligonucleotide synthesis procedures. One approach to overcome this problem is to construct the AO with alternative nucleic acid chemistries using solid-phase oligonucleotide synthesis via standard phosphoramidite chemistry. 2′-Fluoro (2′-F) is a potent RNA analogue that possesses high RNA binding affinity and resistance to nuclease degradation with good safety profile, and an approved drug Macugen containing 2′-F-modified pyrimidines was approved for the treatment of age-related macular degeneration (AMD). In the present study, we investigated the scope of 2′-F nucleotides to construct mixmer and gapmer exon skipping AOs with either 2′-O-methyl (2′-OMe) or locked nucleic acid (LNA) nucleotides on a phosphorothioate (PS) backbone, and evaluated their efficacy in inducing exon-skipping in mdx mouse myotubes in vitro. Our results showed that all AOs containing 2′-F nucleotides induced efficient exon-23 skipping, with LNA/2′-F chimeras achieving better efficiency than the AOs without LNA modification. In addition, LNA/2′-F chimeric AOs demonstrated higher exonuclease stability and lower cytotoxicity than the 2′-OMe/2′-F chimeras. Overall, our findings certainly expand the scope of constructing 2′-F modified AOs in splice modulation by incorporating 2′-OMe and LNA modifications.

Integrin alpha-4 (ITGA4) is a validated therapeutic target for multiple sclerosis (MS) and Natalizumab, an antibody targeting ITGA4 is currently approved for treating MS. However, there are severe side effects related to this therapy. In this study, we report the development of a novel DNAzyme that can efficiently cleave the ITGA4 transcript. We designed a range of DNAzyme candidates across various exons of ITGA4. RNV143, a 30mer arm-loop-arm type DNAzyme efficiently cleaved 84% of the ITGA4 mRNA in human primary fibroblasts. RNV143 was then systematically modified by increasing the arm lengths on both sides of the DNAzymes by one, two and three nucleotides each, and incorporating chemical modifications such as inverted-dT, phosphorothioate backbone and LNA-nucleotides. Increasing the arm length of DNAzyme RNV143 did not improve the efficiency however, an inverted-dT modification provided the most resistance to 3′ → 5′ exonuclease compared to other modifications tested. Our results show that RNV143A could be a potential therapeutic nucleic acid drug molecule towards the treatment for MS.