(P 94) DEVELOPING NOVEL OLIGONUCLEOTIDE THERAPIES TO MODULATE INSULIN RECEPTOR ALTERNATIVE SPLICING IN PEDIATRIC BONE SARCOMAS

Objective: Despite multifaceted treatment approaches for pediatric bone sarcomas, cancer recurrence and relapse are common, and survival outcomes for these young patients are as low as 25%. Both osteosarcoma (OS) and Ewing sarcoma (ES) have harnessed the IGF-I receptor (IGF-IR) and the IGF/insulin receptor (IR) signaling pathways for their progression and spread. Initial anti-IGF-IR therapies like figitumumab were unsuccessful in a majority of patients in part due to the IR, which can bind the same ligands as IGF-IR and potentiate downstream signaling despite IGF-IR inhibition. IR has two alternatively spliced isoforms—IR-A (exon 11 skipped) and IR-B (exon 11 included). Increased expression of IR-A, an isoform frequently expressed in cancer, may effectively evade current therapeutic mechanisms and contribute to resistance in patients.

Our previous work illustrates the development of a splice-switching oligonucleotide (SSO) that significantly shifts IR splicing toward IR-B and slows rhabdomyosarcoma proliferation by targeting the binding sequence of CUG-BP1, a negative splicing regulator upstream of exon 11 (PMID: 35017650). We sought to evaluate the efficacy of this SSO in OS and ES cell lines and patient-derived xenografts (PDX). We also focused on improving in vitro and in vivo delivery of our SSO by expressing it via an adeno-associated viral (AAV) vector.

Methods: We used standard PCR protocols to assess IR isoform expression in a panel of OS and ES cell lines and PDXs at baseline and upon 24 hour SSO transfection with Lipofectamine 3000. We measured OS cell proliferation after transfection of our IR SSO or a non-specific control SSO with the Incucyte system. AAV8-eGFP transduction in OS and ES cells was performed alongside a GFP control plasmid over 72 hours in low-serum media.

Results: We observed a predominant IR-A:IR-B ratio in human OS and ES cell lines, dog OS PDXs, and human ES PDXs. To study this imbalance in IR alternative splicing, we transfected OS and ES cell lines with our 18-mer SSO targeting CUG-BP1 or a non-specific control 18-mer SSO at variable doses. We achieved at least a two-fold increase in IR-B expression in OS cell lines and a modest splicing switch in ES cell lines in a dose-dependent manner. This shift toward IR-B expression due to our SSO also significantly slowed OS cell proliferation as compared to the non-specific SSO.

To address inefficient delivery in ES cells in vitro and OS tumor treatment in vivo, we designed various AAV plasmids to express the IR SSO sequence as an antisense RNA (aRNA) in transduced cells. Since splicing is a nuclear process, we used a promoter derived from the U7-snRNP particle, which has been well characterized in the literature to improve SSO entry into the nucleus and incorporation into the spliceosome to induce splicing changes. We have demonstrated successful transduction of AAV-8 eGFP vectors over a 72-hour time course in ES cells and are currently validating our aRNA-AAV candidate plasmids to identify the best candidate that switches IR splicing from IR-A toward IR-B.

Conclusion: Here, we confirm that OS and ES cells and PDXs preferentially express IR-A and that we can therapeutically modulate IR alternative splicing with our SSO in a dose-dependent manner. Additionally, our preliminary results indicate that AAVs, an FDA-approved methodology to deliver SSOs in a tissue-specific manner in pediatric diseases, are a promising approach for pediatric sarcomas as well. Targeting IR alternative splicing with splice-switching therapies and AAV delivery has the potential to address a key resistance mechanism that rendered previously developed anti-IGF-IR therapies ineffective in pediatric sarcoma patients.