Alpha Omega Alpha Honor Medical Society

2012 Research Abstract

A novel tetanus toxin subfragment as a neurotrophic vector for GM2 gangliosidosis therapy

Investigator: Ryan Lippell, New York Medical College

Mentor: Kostantin Dobrenis, PhD, New York Medical College

More than two-thirds of lysosomal storage diseases (LSDs) feature neuronopathic involvement that results in progressive and ultimately fatal neurological impairment. Enzyme, stem cell and gene replacement therapeutic approaches all face significant difficulties posed by the anatomic and physiologic obstacles of the CNS. In an effort to overcome the blood brain barrier, poor constitutive neuronal endocytosis and need for widespread CNS delivery, we developed and characterized a highly shortened peptide (HcPEP), derived from the C-terminal half of atoxic Hc fragment of tetanus toxin. Studies indicate that HcPEP confers a 30-fold increase in neuronal binding and uptake, as well as lysosomal delivery and retrograde axonal transport to a fluorescent partner protein (protein-HcPEP).

Here we begin translation of the HcPEP-mediated approach to a disease state, utilizing a murine model of Sandhoff disease (SD), a neuronopathic LSD in which the β subunit (hexβ) of β-N-acetylhexosaminidase is deficient and results in absence of both HexA and HexB isozymes. To prepare recombinant Hex-HcPEP enzyme, immortalized hexβ -/- microglia were transduced with lentiviral particles encoding hexβ-HcPEP-IRES-hrGFP. Following verification of secretion, an important pre-requisite for cell-mediated approaches, fluorometric activity assays were performed using HexA and HexB specific substrates. Importantly, HexA-HcPEP was verified to retain HexA activity, indicating that fusion hexβ-HcPEP subunit is successfully combining with native hexα subunit without significant post-translational disruption. Additionally, transduction of hexβ -/- microglia with Hexβ-HcPEP-IRES-hrGFP as compared to Hexβ-IRES-hrGFP led to a statistically significant decrease in the percentage of GM2-storage positive cells (p<0.0001, chi- square), supporting HexA-HcPEP is active against its natural substrate, GM2 ganglioside. Further experiments used WT and SD neuronal cultures prepared from fetal day-15 mouse neocortex to evaluate neuronal uptake, binding and GM2-levels. Ongoing studies are aimed at more fully characterizing recombinant Hex-HcPEP, comparing it to native Hex and purifying both to begin in vivo ERT experiments.

Updated on June 24, 2013.

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