Trastuzumab improves overall survival for HER2+ breast cancer, but its short half-life in the cerebrospinal fluid (~2–4 days) and delivery limitations restrict the ability to target HER2+ central nervous system (CNS) disease. We developed an adeno-associated virus (AAV) vector expressing a codon-optimized, ubiquitin C (UbC)-promoter-driven trastuzumab sequence (AAV9.UbC.trastuzumab) for intrathecal administration. Transgene expression was evaluated in adult Rag1 knockout mice and rhesus nonhuman primates (NHPs) after a single intracerebroventricular (ICV) or intra-cisterna magna (ICM) AAV9.UbC.trastuzumab injection, respectively, using real-time PCR, ELISA, Western blot, in situ hybridization, single-nucleus RNA sequencing, and liquid chromatography-mass spectrometry; antitumor efficacy was evaluated in brain xenografts using HER2+ breast cancer cell lines (BT-474, MDA-MB-453). Transgene expression was detected in brain homogenates of Rag1 knockout mice following a single ICV injection of AAV9.UbC.trastuzumab (1 × 10¹¹ vector genome copies [GC]/mouse) and tumor progression was inhibited in xenograft models of breast-to-brain metastasis. In NHPs, ICM delivery of AAV9.UbC.trastuzumab (3 × 10¹³ GC/animal) was well tolerated (36–37 days in-life) and resulted in transgene expression in CNS tissues and cerebrospinal fluid at levels sufficient to induce complete tumor remission in MDA-MB-453 brain xenografts. With AAV9’s proven clinical safety record, this gene therapy may represent a viable approach for targeting HER2 + CNS malignancies.

曲妥珠单抗可改善 HER2+ 乳腺癌的总体生存率，但其在脑脊液中的半衰期较短（约 2-4 天）和递送限制限制了针对 HER2+ 中枢神经系统 (CNS) 疾病的能力。我们开发了一种腺相关病毒 (AAV) 载体，表达密码子优化、泛素 C (UbC) 启动子驱动的曲妥珠单抗序列 (AAV9.UbC.曲妥珠单抗)，用于鞘内给药。使用实时 PCR、ELISA、Western blot 分别在脑室内 (ICV) 或小脑延髓池内 (ICM) AAV9.UbC.trastuzumab 注射后，评估成年Rag1敲除小鼠和恒河非人灵长类动物 (NHP) 中的转基因表达、原位杂交、单核RNA测序和液相色谱-质谱联用；使用 HER2+ 乳腺癌细胞系（BT-474、MDA-MB-453）在脑异种移植物中评估抗肿瘤功效。单次 ICV 注射 AAV9.UbC.trastuzumab（1 × 10 ^{11载体基因组拷贝 [GC]/小鼠）后，在}Rag1敲除小鼠的脑匀浆中检测到转基因表达，并且在乳脑异种移植模型中肿瘤进展受到抑制转移。^{在 NHP 中，AAV9.UbC.曲妥珠单抗（3 × 10 13} GC/动物）的 ICM 递送耐受性良好（生命中 36-37 天），并导致中枢神经系统组织和脑脊液中的转基因表达水平足以诱导完全肿瘤MDA-MB-453 脑异种移植物的缓解。凭借 AAV9 经证实的临床安全记录，这种基因疗法可能代表一种针对 HER2 + CNS 恶性肿瘤的可行方法。