Engineering Brain Parasites for Intracellular Delivery of Therapeutic Proteins

Abstract

Protein therapy has the potential to alleviate many neurological diseases; however, 15 delivery mechanisms for the central nervous system (CNS) are limited, and intracellular delivery poses additional hurdles. To address these challenges, we harnessed the protist parasite Toxoplasma gondii, which can migrate into the CNS and secrete proteins into cells. Using a fusion protein approach, we engineered T. gondii to secrete therapeutic proteins for human neurological disorders. We tested two secretion systems, generated fusion proteins that localized 20 to T. gondii's secretory organelles and assessed their intracellular targeting in various mammalian cells including neurons. We show that T. gondii expressing GRA16 fused to the Rett syndrome protein MeCP2 deliver a fusion protein that mimics the endogenous MeCP2, binding heterochromatic DNA in neurons. This demonstrates the potential of T. gondii as a therapeutic protein vector, which could provide either transient or chronic, in situ synthesis and delivery of 25 intracellular proteins to the CNS. 45

IFN-γ stimulated murine and human neurons mount anti-parasitic defenses against the intracellular parasiteToxoplasma gondii

“…Compared to II-RCre parasites, II-GCre parasites showed a decreased efficiency of causing Cre-mediated recombination (>90% vs. 50%, Fig. S3B), which was expected because host cell-exported GRA proteins show decreased exportation when fused to ordered proteins (Bracha et al, 2018;Curt-Varesano et al, 2016;Franco et al, 2016).…”

Quantifying and contextualizing the impact of bioRxiv preprints through automated social media audience segmentation

“…For example, the preprint “DeepPoseKit, a software toolkit for fast and robust animal pose estimation using deep learning,” by Graving and colleagues, was submitted to bioRxiv in the “animal behavior and cognition” category [ 36 ], and topics 1 and 12 included the keyword “animal,” indicating a match for this focal category (also note that topics 1 and 12 for this preprint included the keyword “ecology”) ( Fig 1A ). “Engineering Brain Parasites for Intracellular Delivery of Therapeutic Proteins,” by Bracha and colleagues, was submitted to bioRxiv in the “synthetic biology” category [ 37 ]—though we did not identify any topics containing the keyword “synthetic,” several topics indicated a match with academic disciplines that appear related to this subject, including neuroscience (topic 5) and medical research (topics 11 and 12) ( Fig 1B ). “Sex differences in gene expression in the human fetal brain,” by O’Brien and colleagues, was submitted to bioRxiv in the “genomics” category [ 38 ], with topic 1 indicating a match with the focal category ( Fig 1C ).…”

“…"DeepPoseKit, a software toolkit for fast and robust animal pose estimation using deep learning," by Graving and colleagues [36], included nonacademic audience sectors associated with video game developers (topic 2), business applications of artificial intelligence (topics 6 and 9), and graphic designers (topic 11) (Fig 1A), presumably because this research has implications for more realistic computational rendering of physiological properties and organic movement. "Engineering Brain Parasites for Intracellular Delivery of Therapeutic Proteins," by Bracha and colleagues [37], included nonacademic audience sectors we ascribe to be primarily associated with biotechnology companies (topics 2, 8, 9, and 10), aligning with the paper's focus on bioengineering, as well as an audience sector associated with news media (topic 3), suggesting this preprint caught the attention of science journalists (though we note that at the time we analyzed these data, Altmetric has not indexed any news media coverage that cites this preprint) ( Fig 1B). Other preprints showed a greater variety of nonacademic audience sectors.…”

IFN-γ stimulated murine and human neurons mount anti-parasitic defenses against the intracellular parasite Toxoplasma gondii

“…Compared to II-RCre parasites, II-GCre parasites were less efficient at triggering Cre-mediated recombination (>90 vs. 50%, Supplementary Fig. 3B), which was expected because host cell-exported GRA proteins show decreased exportation when fused to ordered proteins 41,44,45 .…”