Perdigão Pedro R L, Cunha-Santos Catarina, Barbas Carlos F, Santa-Marta Mariana, Goncalves Joao
Molecular Microbiology and Biotechnology Department, Research Institute for Medicines (iMed ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal.
Department of Chemistry, Department of Cell and Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, USA.
Mol Ther Methods Clin Dev. 2020 May 22;18:145-158. doi: 10.1016/j.omtm.2020.05.016. eCollection 2020 Sep 11.
Despite efforts to develop effective treatments for eradicating HIV-1, a cure has not yet been achieved. Whereas antiretroviral drugs target an actively replicating virus, latent, nonreplicative forms persist during treatment. Pharmacological strategies that reactivate latent HIV-1 and expose cellular reservoirs to antiretroviral therapy and the host immune system have, so far, been unsuccessful, often triggering severe side effects, mainly due to systemic immune activation. Here, we present an alternative approach for stimulating latent HIV-1 expression via direct protein delivery of cell-penetrating zinc-finger activators (ZFAs). Cys-His zinc-fingers, fused to a transcription activation domain, were engineered to recognize the HIV-1 promoter and induce targeted viral transcription. Following conjugation with multiple positively charged nuclear localization signal (NLS) repeats, protein delivery of a single ZFA (3NLS-PBS1-VP64) efficiently internalized HIV-1 latently infected T-lymphocytes and specifically stimulated viral expression. We show that short-term treatment with this ZFA protein induces higher levels of viral reactivation in cell line models of HIV-1 latency than those observed with gene delivery. Our work establishes protein delivery of ZFA as a novel and safe approach toward eradication of HIV-1 reservoirs.
尽管人们努力研发有效的治疗方法来根除HIV-1，但尚未实现治愈。抗逆转录病毒药物针对的是活跃复制的病毒，而潜伏的、非复制形式的病毒在治疗期间持续存在。到目前为止，重新激活潜伏的HIV-1并使细胞储存库暴露于抗逆转录病毒疗法和宿主免疫系统的药理学策略尚未成功，常常引发严重的副作用，主要是由于全身免疫激活。在此，我们提出一种通过直接蛋白质递送细胞穿透性锌指激活剂（ZFA）来刺激潜伏HIV-1表达的替代方法。与转录激活结构域融合的半胱氨酸-组氨酸锌指被设计用于识别HIV-1启动子并诱导靶向病毒转录。与多个带正电荷的核定位信号（NLS）重复序列缀合后，单一ZFA（3NLS-PBS1-VP64）的蛋白质递送有效地内化了潜伏感染HIV-1的T淋巴细胞并特异性刺激了病毒表达。我们表明，与基因递送相比，用这种ZFA蛋白进行短期治疗在HIV-1潜伏的细胞系模型中诱导出更高水平的病毒重新激活。我们的工作确立了ZFA的蛋白质递送作为一种根除HIV-1储存库的新颖且安全的方法。
