 Hi, my name is Rachita Sumbria and I am presenting a video abstract for the article titled Pharmapokinetics and Brain Uptakes in the Renaissance Monty of Occlusion Protein of Arryl Sulphatase A and a Monoclonal Antibody against the human insulin receptor. So to begin with a brief introduction, Metachromatic Neucodistrophy or MLD is a devastating lysosomal storage disorder first due to the deficiency or loss of Arryl Sulphatase A enzyme. Arryl Sulphatase A is responsible for the degradation of sphingolipid Sulphatase and pathological attrumination of these Sulphatase leads to demyelination causing severe CNS disturbances depending on the type of MLD. In the most common form of MLD that is infantile MLD, children suffer from severe CNS abnormalities like dementia, blindness, convulsions and eventually die before the age of 5. Replacement of the missing Arryl Sulphatase A enzyme thus represents a very promising approach for the treatment of MLD. However, enzyme replacement therapy has not been successful for the CNS since Arryl Sulphatase A does not cross the blood-brain barrier. Intrathical injection of Arryl Sulphatase A has been used to bypass the blood-brain barrier. However, this route leads to distribution only to the ependymal surface of the brain. Penetration of the drug from the CSF to the brain is further limited by diffusion which is slow due to rapid bulk flow from CSF to the blood. Arryl Sulphatase A can however be delivered via the transvascular route by re-engineering the enzyme for blood-brain barrier penetration using the molecular torsion horse technology. In the present study, a fusion protein of Arryl Sulphatase A and a monoclonal antibody against the human insulin receptor is engineered. The fusion protein is designated as HIRMABASA and is shown in the right panel. The HIRMAB domain of the fusion protein binds to endogenous insulin receptors and crosses the blood-brain barrier via receptor-mediated transverse, thus acting as a molecular torsion horse to ferry the ASA or the Arryl Sulphatase A into the brain from the blood. The present study shows that the fusion protein retains high affinity binding to the human insulin receptor with an EC50 of 0.3 nanomoners and also retains high Arryl Sulphatase A enzyme activity of 20 units per milligram. In order to further characterize the plasma pharmacokinetic and the brain uptake of the fusion protein in Vivo, the fusion protein was radioiodinated and injected intravenously in Rehesis monkey. In the present study, the fusion protein was radioiodinated using the Bolton Hunter reagent. As opposed to the classic oxidative radiolabeling methods like chloromint T and iodogen that are commonly used, the Bolton Hunter radiolabeling is non-oxidative, thus less damaging to the protein. Oxidative methods iodinate tyrosine residues and peripheral degradation of the protein radiolabelled with oxidative methods release iodotyrosine that is transported across the blood-brain barrier via amino acid transporters. This leads to high radioactivity in the brain that in fact represents radio-enabled metabolites and not the impact protein. Bolton Hunter reagent, on the other hand, iodinate epsilon-amino group of surface lysine residues. Blood-borne Bolton Hunter conjugated lysine residues are not transported across the blood-brain barrier. After intravenous administration, iodinated fusion protein rapidly penetrates the Rehesis monkey brain with a brain uptake of 1.1% injected dose for brain and this uptake is comparable to the brain uptake of phalloprite which is a lipid soluble small molecule. Film autoreliography shows global distribution of the fusion protein with higher uptake in the grey matter as compared to the white matter. Penetration of the fusion protein in the brain parenchroma is confirmed in the Prasmsali by capillary depletion method and emulsion autoreliography of the monkey brain. Emulsion autoreliography shows silver grains in the brain micro-vessels and uniform distribution throughout the brain parenchroma. While the TCA precipitability of the fusion protein is only 42% in the plasma at 120 minutes the TCA precipitability of the fusion protein in the brain is 95% confirming that the silver grains as seen in the emulsion autoreliography represents intact fusion protein and not radiolabel metabolite. Finally, when metachromatic leukodystrophies fibroblast cells were incubated with the fusion protein for 24 hours and cells prepared for dual-labeled confocal microscopy for lysosomal compartment and human aryl sulfatase A we found sequestration of the fusion protein in the lysosomal compartment. Once in the lysosome the aryl sulfatase A enzyme domain of the fusion protein is active. Hence the Prasmsali concludes that the HIRMAV aryl sulfatase A fusion protein is a new biological entity for the treatment of the brain in humans with metachromatic leukodystrophy following non-invasive intravenous infusions. For further details about the studies please refer to the online published article. Thank you and I hope you enjoy reading.