Tetanus toxin fragment C fusion facilitates protein delivery to CNS neurons from cerebrospinal fluid in mice

Authors

  • Susanna C. Benn,

    1. Cecil B. Day Laboratory for Neuromuscular Research, Department of Neurology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
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      Susanna C. Benn and Ilknur Ay contributed equally to this work.

  • Ilknur Ay,

    1. Cecil B. Day Laboratory for Neuromuscular Research, Department of Neurology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
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      Susanna C. Benn and Ilknur Ay contributed equally to this work.

  • Elena Bastia,

    1. Laboratory of Molecular Neurobiology, Department of Neurology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
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  • Ru-Ju Chian,

    1. Cecil B. Day Laboratory for Neuromuscular Research, Department of Neurology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
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  • Samuel A. Celia,

    1. Cecil B. Day Laboratory for Neuromuscular Research, Department of Neurology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
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  • R. Blake Pepinsky,

    1. BiogenIdec, Inc., Cambridge, Massachusetts, USA
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  • Paul S. Fishman,

    1. Research Service, Baltimore Veterans Affairs Medical Center, Baltimore, Maryland, USA
    2. Department of Neurology, University of Maryland School of Medicine, Baltimore, Maryland, USA
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  • Robert H. Brown Jr,

    1. Cecil B. Day Laboratory for Neuromuscular Research, Department of Neurology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
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  • Jonathan W. Francis

    1. Cecil B. Day Laboratory for Neuromuscular Research, Department of Neurology, Massachusetts General Hospital, Charlestown, Massachusetts, USA
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Address correspondence and reprint requests to Jonathan W. Francis, Cecil B. Day Laboratory for Neuromuscular Research, Massachusetts General Hospital, Building 114, 16th Street, Room 3003, Charlestown, MA 02129, USA. E-mail: jwfrancis@partners.org

Abstract

To improve protein delivery to the CNS following intracerebroventricular administration, we compared the distribution of a human Cu/Zn superoxide dismutase:tetanus toxin fragment C fusion protein (SOD1:TTC) in mouse brain and spinal cord with that of tetanus toxin fragment C (TTC) or human SOD1 (hSOD1) alone, following continuous infusion into the lateral ventricle. Mice infused with TTC or SOD1:TTC showed intense anti-TTC or anti-hSOD1 labeling, respectively, throughout the CNS. In contrast, animals treated with hSOD1 revealed moderate staining in periventricular tissues. In spinal cord sections from animals infused with SOD1:TTC, the fusion protein was found in neuron nuclear antigen-positive (NeuN+) neurons and not glial fibrillary acidic protein-positive (GFAP+) astrocytes. The percentage of NeuN+ ventral horn cells that were co-labeled with hSOD1 antibody was greater in mice treated with SOD1:TTC (cervical cord = 73 ± 8.5%; lumbar cord = 62 ± 7.7%) than in mice treated with hSOD1 alone (cervical cord = 15 ± 3.9%; lumbar cord = 27 ±4.7%). Enzyme-linked immunosorbent assay for hSOD1 further demonstrated that SOD1:TTC-infused mice had higher levels of immunoreactive hSOD1 in CNS tissue extracts than hSOD1-infused mice. Following 24 h of drug washout, tissue extracts from SOD1:TTC-treated mice still contained substantial amounts of hSOD1, while extracts from hSOD1-treated mice lacked detectable hSOD1. Immunoprecipitation of SOD1:TTC from these extracts using anti-TTC antibody revealed that the recovered fusion protein was structurally intact and enzymatically active. These results indicate that TTC may serve as a useful prototype for development as a non-viral vehicle for improving delivery of therapeutic proteins to the CNS.

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