37P - Development of splice switching antisense oligonucleotides targeting midkine

Background

Midkine is a multi-functional growth factor/cytokine that is involved in diverse solid and haematological cancers. Midkine mediates critical cell interactions within the tumour microenvironment, and thereby contributes to metastasis (Nature 2017), immunosuppression and resistance to immune checkpoint inhibitors (Nature Medicine 2020; Nature Comms 2022) and angiogenesis. Therefore, midkine may be a novel target, particularly in hard to treat or resistant tumours.While various biologicals inhibit midkine in animal models of cancer, splice switching antisense oligonucleotides (SSOs) have not been evaluated. SSOs uniquely reduce the levels of full length midkine protein and also generate non-functional, truncated midkine due to exon skipping.

Methods

Guided by SpliceAid to identify splice enhancer binding motifs SSOs were designed to predicted splice motifs in exons 3 and 4 of the midkine mRNA and synthesized using 2’OMe-PS nucleotide chemistry. Exon skipping was initially assessed by RT-PCR with primers flanking exons 2 and 5.

Results

Transfection into midkine-expressing human Huh7 liver and SHSY5Y neuroblastoma cancer cells elicited up to 30% of mRNA missing the targeted exons. Optimisation of lead SSOs through microwalking, cocktails of SSOs and PMO chemistry resulted in >90% exon skipped midkine. Importantly, the truncated midkine protein, corresponding to the shorter mRNA lacking Exon 4, was produced by cancer cells. Studies are underway to examine the functional outcomes of midkine SSOs on cancer cell proliferation/apoptosis, migration, invasion and angiogenesis.

Conclusions

Lead midkine SSOs will then be assessed for their ability to alter in vivo tumour growth and metastasis as a prelude for further pre-clinical development.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

Lyramid Ltd.

Funding

Lyramid Ltd.

Disclosure

G.R. Robertson: Financial Interests, Personal, Full or part-time Employment: Lyramid Ltd. All other authors have declared no conflicts of interest.