Department of Nuclear Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802, United States.
Department of Pediatrics, Centre of Blood Oxygen Transport & Hemostasis, University of Maryland-Baltimore School of Medicine, Baltimore, Maryland 21201, United States.
ACS Nano. 2024 Mar 26;18(12):9199-9220. doi: 10.1021/acsnano.4c01400. Epub 2024 Mar 11.
The majority of triple negative breast cancers (TNBCs) are basal-like breast cancers (BLBCs), which tend to be more aggressive, proliferate rapidly, and have poor clinical outcomes. A key prognostic biomarker and regulator of BLBC is the Forkhead box C1 (FOXC1) transcription factor. However, because of its functional placement inside the cell nucleus and its structural similarity with other related proteins, targeting FOXC1 for therapeutic benefit, particularly for BLBC, continues to be difficult. We envision targeted nonviral delivery of CRISPR/Cas9 plasmid toward the efficacious knockdown of FOXC1. Keeping in mind the challenges associated with the use of CRISPR/Cas9 in vivo, including off-targeting modifications, and effective release of the cargo, a nanoparticle with context responsive properties can be designed for efficient targeted delivery of CRISPR/Cas9 plasmid. Consequently, we have designed, synthesized, and characterized a zwitterionic amino phospholipid-derived transfecting nanoparticle for delivery of CRISPR/Cas9. The construct becomes positively charged only at low pH, which encourages membrane instability and makes it easier for nanoparticles to exit endosomes. This has enabled effective in vitro and in vivo downregulation of protein expression and genome editing. Following this, we have used EpCAM aptamer to make the system targeted toward BLBC cell lines and to reduce its off-target toxicity. The in vivo efficacy, biodistribution, preliminary pharmacokinetics, and biosafety of the optimized targeted CRISPR nanoplatform is then validated in a rodent xenograft model. Overall, we have attempted to knockout the proto-oncogenic FOXC1 expression in BLBC cases by efficient delivery of CRISPR effectors via a context-responsive nanoparticle delivery system derived from a designer lipid derivative. We believe that the nonviral approach for in vitro and in vivo delivery of CRISPR/Cas9 targeted toward FOXC1, studied herein, will greatly emphasize the therapeutic regimen for BLBC.
大多数三阴性乳腺癌（TNBC）是基底样乳腺癌（BLBC），它们往往更具侵袭性，增殖迅速，临床预后较差。FOXC1 转录因子是 BLBC 的关键预后生物标志物和调节因子。然而，由于其在细胞核内的功能位置及其与其他相关蛋白的结构相似，针对 FOXC1 进行治疗获益，特别是针对 BLBC，仍然具有挑战性。我们设想通过靶向非病毒递送 CRISPR/Cas9 质粒，实现 FOXC1 的有效敲低。考虑到 CRISPR/Cas9 在体内应用的挑战，包括脱靶修饰和有效释放货物，我们可以设计具有上下文响应特性的纳米颗粒，以实现高效靶向递送 CRISPR/Cas9 质粒。因此，我们设计、合成并表征了一种两性离子氨基磷脂衍生的转染纳米颗粒，用于递送 CRISPR/Cas9。该构建体仅在低 pH 值下带正电荷，这会促使膜不稳定，并使纳米颗粒更容易从内体中逸出。这使得在体外和体内有效下调蛋白表达和基因组编辑成为可能。在此之后，我们使用 EpCAM 适体使该系统靶向 BLBC 细胞系，并降低其脱靶毒性。然后，在啮齿动物异种移植模型中验证了优化的靶向 CRISPR 纳米平台的体内功效、生物分布、初步药代动力学和生物安全性。总的来说，我们试图通过从设计的脂质衍生物衍生的上下文响应纳米颗粒递送系统，高效递送至 BLBC 病例中的原癌基因 FOXC1，从而敲除其表达。我们相信，本文研究的针对 FOXC1 的 CRISPR 效应物的体外和体内非病毒递送方法，将极大地强调 BLBC 的治疗方案。
