VEGF suppression via exon 2 splice-switching Vivo-Morpholino antisense oligo to inhibit tumor growth in a mouse subcutaneous model.

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Background: VEGF-A is a central driver of tumor angiogenesis and is expressed as multiple pro-angiogenic splice isoforms. Current anti-VEGF therapies neutralize extracellular VEGF protein but do not reduce VEGF production at the mRNA level or address alternative splicing. To achieve pan-isoform suppression, we developed a splice-switching Vivo-Morpholino targeting the VEGF exon 2 splice junction, a sequence common to all known VEGF isoforms. Inducing exon 2 skipping creates a downstream frameshift, leading to reduced total VEGF expression. Methods: C57BL/6 mice were inoculated subcutaneously with Lewis lung carcinoma cells. Once tumors were palpable, animals were randomized to receive a Vivo-Morpholino antisense oligonucleotide targeting the mouse VEGF exon 2 splice junction. For systemic delivery, mice received intravenous injections at 15 mg/kg twice weekly for two to three weeks. For local delivery, mice received intratumoral injections of 0.24 mg once weekly for two to three weeks. Mice were sacrificed one week after the final IV dose. VEGF Exon 2 skipping were assessed in tumor tissue and major organs by RT-PCR. Tumor growth was monitored by caliper measurements, and volumes were calculated using standard formulas. Results: Treatment induced VEGF exon 2 skipping in tumors and in various tissues. After IV administration, knockdown reached 10% in s.c. tumor tissue, 90% in kidney, and 95% in liver, demonstrating effective systemic delivery with higher uptake in well-perfused organs. Despite modest tumor knockdown following IV dosing, tumor growth was significantly inhibited, with an 83% reduction in tumor volume compared with controls. Direct intratumoral injection reduced tumor volume by 71%. These data indicate that partial suppression of tumor-derived VEGF is sufficient to produce substantial anti-tumor effects in this model. Conclusions: Splice-switching of VEGF exon 2 using a Vivo-Morpholino enables pan-isoform VEGF suppression through frameshift-mediated knockdown. Both systemic and intratumoral delivery significantly inhibited tumor growth in a mouse subcutaneous model. This upstream RNA-targeting strategy represents a novel anti-angiogenic approach that warrants further development for cancer therapy.