Amplification of plays a pivotal role in multiple types of tumors and correlates with poor prognosis in high-risk neuroblastoma. Despite recent advances in the treatment of neuroblastoma, no approaches directly target the master oncogene . Difficulties in targeting the MYCN protein inspired us to develop a new gene-level-inhibitory strategy using a sequence-specific gene regulator. Here, we generated a -targeting pyrrole-imidazole (PI) polyamide, MYCN-A3, which directly binds to and alkylates DNA at homing motifs within the transcript. Pharmacologic suppression of MYCN inhibited the proliferation of cancer cells harboring amplification compared with nonamplified cancer cells. In neuroblastoma xenograft mouse models, MYCN-A3 specifically downregulated MYCN expression and suppressed tumor progression with no detectable adverse effects and resulted in prolonged overall survival. Moreover, treatment with MYCN-A3, but not nontargeting PI polyamide, precipitated a copy number reduction of in neuroblastoma cells with amplification. These findings suggest that directly targeting with MYCN-A3 is a novel therapeutic approach to reduce copy number of the gene for -amplified neuroblastoma. SIGNIFICANCE: This study presents a novel approach to drugging an amplified oncogene by showing that targeting gene amplification of suppresses MYCN expression and neuroblastoma growth.