Engineering Toxoplasma gondii secretion systems for intracellular delivery of multiple large therapeutic proteins to neurons.
Shahar BrachaHannah J JohnsonNicole A PranckeviciusFrancesca CattoAthena E EconomidesSergey LitvinovKaroliina HassiMarco Tullio RigoliCristina CheroniMatteo BonfantiAlessia ValentiSarah StucchiShruti AttreyaPaul D RossDaniel WalshNati MalachiHagay LivneReut EshelVladislav KrupalnikDoron LevinStuart CobbPetros KoumoutsakosNicolò CaporaleGiuseppe TestaAdriano AguzziAnita A KoshyLilach SheinerOded RechaviPublished in: Nature microbiology (2024)
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.
Keyphrases
- toxoplasma gondii
- induced apoptosis
- resting state
- white matter
- endothelial cells
- cell cycle arrest
- spinal cord
- functional connectivity
- quantum dots
- high resolution
- type diabetes
- skeletal muscle
- oxidative stress
- endoplasmic reticulum stress
- high throughput
- amino acid
- multiple sclerosis
- signaling pathway
- mass spectrometry
- blood brain barrier
- cell death
- current status
- case report
- cerebrospinal fluid
- pi k akt
- gene expression