跨越血脑屏障等生物屏障递送大分子限制了它们在体内的应用。先前的研究表明，弓形虫是一种从人体肠道自然传播到中枢神经系统（CNS）的寄生虫，可以将蛋白质输送到宿主细胞。在这里，我们设计了弓形虫的内源性分泌系统，即菱形和致密颗粒，通过与 toxofilin 和 GRA16 的翻译融合，将多种大的 (>100kDa) 治疗蛋白递送到神经元中。我们展示了在培养细胞、脑类器官和体内的递送，并使用成像、pull-down 测定、scRNA-seq 和荧光报告基因探测蛋白质活性。我们证明了小鼠腹腔给药后的稳健递送，并表征了整个大脑的 3D 分布。作为概念证明，我们证明了 GRA16 介导的 MeCP2 蛋白的脑传递，这是雷特综合征的假定治疗靶点。通过描述系统的潜力和当前局限性，我们的目标是指导未来更广泛应用所需的改进。© 2024。作者。

Delivering macromolecules across biological barriers such as the blood-brain barrier limits their application in vivo. Previous work has demonstrated that Toxoplasma gondii, a parasite that naturally travels from the human gut to the central nervous system (CNS), can deliver proteins to host cells. Here we engineered T. gondii's endogenous secretion systems, the rhoptries and dense granules, to deliver multiple large (>100 kDa) therapeutic proteins into neurons via translational fusions to toxofilin and GRA16. We demonstrate delivery in cultured cells, brain organoids and in vivo, and probe protein activity using imaging, pull-down assays, scRNA-seq and fluorescent reporters. We demonstrate robust delivery after intraperitoneal administration in mice and characterize 3D distribution throughout the brain. As proof of concept, we demonstrate GRA16-mediated brain delivery of the MeCP2 protein, a putative therapeutic target for Rett syndrome. By characterizing the potential and current limitations of the system, we aim to guide future improvements that will be required for broader application.© 2024. The Author(s).