Antisense oligonucleotide induced therapeutic alternative splicing: A flexible platform capable of addressing inherited and acquired diseases

Bal Hari H Poudel

Introduction Antisense oligonucleotides (AOs) are synthetic nucleic acids that can anneal to target RNA or DNA sequences through Watson and Crick base pairing and modify gene expression. Of the several mechanisms that the AO can exert, my research focuses on manipulating pre-mRNA splicing. To date, six splice manipulating AOs have been approved by the US Food and Drug administration for various diseases, and three were developed in our laboratory. We are now expanding the therapeutic alternative splicing platform to many other diseases. My research focuses on demonstrating the flexibility of this platform to treating amenable dysferlin mutations causing limb girdle muscular dystrophy -LGMD2B, and through ACE2 isoform switching to increase host resistance to COVID-19, a viral infection that has devasted the world health system and economy. AO-mediated skipping of dysferlin exons Dysferlin, encoded by the DYSF gene, is a calcium-dependent membrane-associated protein involved in membrane repair, vesicle trafficking, and T-tubule function. Mutations in DYSF cause primary dysferlinopathy, and more than 500 pathogenic mutations in DYSF have been reported. Currently, there are no drugs available to treat dysferlinopathy. It was shown that dysferlin exon 32 could be bypassed and restore some dysferlin function, and hence we hypothesize that AO-mediated exon-skipping strategy could be applied to some dysferlin mutations. We received cells from a patient with nonsense mutations in dysferlin exons 32 and 52. AOs were designed to induce the skipping of those mutated exons. For their therapeutic potential, the most efficient exon 32, 51 and 52 skipping AOs were assessed in induced myogenic cells derived from patient cells individually and as combinations. Exon 32 skipping AOs and combinations of AOs targeting exons 51 and 52 slightly improved the dysferlin function. Subsequently, additional in-frame exons 25, 30, 34 and 37 which could be possible targets were selected and skipped in healthy donor-derived myogenic cells. Compounds shown to induce some exon skipping were further evaluated as peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO), an AO chemistry showing great clinical promise. I could show that all these target exons can be skipped efficiently as demonstrated by transcript analysis through RT-PCR. Of interest was the observation that exons 25 and 37 AOs were able to produce similar levels of protein as observed in untreated cells. ACE2 isoform switching with antisense oligos However, during the course of my studies, COVID-19 became a house-hold word as the world was gripped by the pandemic. While obvious therapeutic strategies rely on the development of safe and effective vaccines or anti-virals, a collaboration with researchers at Monash University resulted in a novel approach to transiently modify gene expression to increase host resistance to the virus. Angiotensin converting enzyme 2 (ACE2) is highly expressed on the surface of human lung epithelial cells (and many other cell types) and is used by coronaviruses as the primary attachment site to enter human cells. The ACE2 protein can also exist as a catalytically active soluble isoform, following alternative splicing to remove the exon(s) encoding the transmembrane domain near the carboxyl terminus. This soluble isoform is shed into the circulation and recent studies have shown that engineered protein isoforms can act as soluble viral decoys. Hence, using splice switching AOs to redirect ACE2 expression towards the soluble isoform, while simultaneously lowering the membrane-bound isoform should offer a most effective dual approach to reduce COVID-19 infection. Optimal AOs to excise exon 18, encoding the transmembrane domain of ACE2, and also compounds to disrupt expression by skipping exons 15, 16, and 17 were identified. This work was performed in collaboration with Monash University, where all mouse studies were performed and are still in progress, and a significant increase in soluble ACE2 was observed after treatments with exon 18 AO. Conclusion In conclusion, I have designed AOs to induce dysferlin isoforms and shown that efficient exon skipping can be achieved. However, the therapeutic potential of this exon skipping has yet to be confirmed and validated, with a slightly detectable increase in dysferlin function could be demonstrated in the available patient’s cells. On a much more promising note, several AOs have been developed to redirect ACE2 expression from the membrane-bound to the soluble isoform. Preliminary experiments using in vitro cell models of COVID-19 infection have shown a strong reduction in viral load in treated cells compared to untreated cells.

Antisense oligonucleotide induced therapeutic alternative splicing: A flexible platform capable of addressing inherited and acquired diseases

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