Engineering Toxoplasma gondii secretion systems for intracellular delivery of multiple large therapeutic proteins to neurons PMID: 39075233

ADC Targets in Oncology

2026-04-10

Engineering Toxoplasma gondii secretion systems for intracellular delivery of multiple large therapeutic proteins to neurons

Nat Microbiol. 2024 Aug;9(8):2051-2072. doi: 10.1038/s41564-024-01750-6.

Shahar Bracha ¹ ² , Hannah J Johnson ³ ⁴ , Nicole A Pranckevicius ⁵ , Francesca Catto ⁶ , Athena E Economides ⁶ , Sergey Litvinov ⁷ , Karoliina Hassi ⁵ , Marco Tullio Rigoli ⁸ ⁹ ¹⁰ , Cristina Cheroni ⁸ ¹⁰ , Matteo Bonfanti ⁸ , Alessia Valenti ⁸ ⁹ ¹⁰ , Sarah Stucchi ⁸ ⁹ ¹⁰ , Shruti Attreya ¹¹ , Paul D Ross ¹² ¹³ , Daniel Walsh ⁵ , Nati Malachi ¹⁴ , Hagay Livne ¹⁴ , Reut Eshel ¹⁴ , Vladislav Krupalnik ¹⁴ , Doron Levin ¹⁴ , Stuart Cobb ¹² ¹³ , Petros Koumoutsakos ⁷ , Nicolò Caporale ⁸ ⁹ ¹⁰ , Giuseppe Testa ¹⁵ ¹⁶ ¹⁷ , Adriano Aguzzi ¹⁸ , Anita A Koshy ¹⁹ , Lilach Sheiner ²⁰ , Oded Rechavi ²¹

Affiliations

1. Department of Neurobiology, Biochemistry and Biophysics, Wise Faculty of Life Sciences and Sagol School for Neuroscience, Tel Aviv University, Tel Aviv, Israel. [email protected].
2. McGovern Institute for Brain Research, MIT, Cambridge, MA, USA. [email protected].
3. Neuroscience Graduate Interdisciplinary Program, University of Arizona, Tucson, AZ, USA.
4. Departments of Neurology and Immunobiology, College of Medicine, and BIO5 Institute, University of Arizona, Tucson, AZ, USA.
5. Centre for Parasitology, School of Infection and Immunity, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.
6. Institute of Neuropathology, University Hospital Zurich, University of Zurich, Zurich, Switzerland.
7. Computational Science and Engineering Laboratory, School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA.
8. Human Technopole, Milan, Italy.
9. Department of Experimental Oncology, European Institute of Oncology IRCCS, Milan, Italy.
10. Department of Oncology and Hemato-oncology, University of Milan, Milan, Italy.
11. Undergraduate Biology Research Program, University of Arizona, Tucson, AZ, USA.
12. Institute of Neuroscience and Psychology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.
13. Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, UK.
14. Epeius Pharma, Ness Ziona, Israel.
15. Human Technopole, Milan, Italy. [email protected].
16. Department of Experimental Oncology, European Institute of Oncology IRCCS, Milan, Italy. [email protected].
17. Department of Oncology and Hemato-oncology, University of Milan, Milan, Italy. [email protected].
18. Institute of Neuropathology, University Hospital Zurich, University of Zurich, Zurich, Switzerland. [email protected].
19. Departments of Neurology and Immunobiology, College of Medicine, and BIO5 Institute, University of Arizona, Tucson, AZ, USA. [email protected].
20. Centre for Parasitology, School of Infection and Immunity, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK. [email protected].
21. Department of Neurobiology, Biochemistry and Biophysics, Wise Faculty of Life Sciences and Sagol School for Neuroscience, Tel Aviv University, Tel Aviv, Israel. [email protected].

Abstract

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.