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Grant Abstract

Grant Number: 5R01NS053808-02
PI Name: NEUFELD, ELIZABETH F.
Project Title: Aptamer-directed crossing of the blood barrier for enzyme therapy of LSDs

Abstract: DESCRIPTION (provided by applicant): Lysosomal storage disorders (LSDs) are genetic diseases, individually rare, but in the aggregate presenting a serious medical and human burden. Enzyme replacement therapy, anticipated for decades, became a reality for Gaucher disease in the 1990s, for MPS I and Fabry disease in 2003, and is currently in clinical trial for several other LSDs. And yet most LSD patients are unable to benefit from these advances because their disease manifests itself, in whole or in part, in the central nervous system. The administered enzyme does not enter the CNS because of the blood brain barrier (BBB) created by the brain microvasculature. However, there exist physiological systems for transporting proteins needed by the brain. We propose to utilize the transcytosis of the transferrin receptor (TfR) to ferry therapeutic enzymes across the BBB. Binding to the TfR will be mediated by aptamers selected for binding to that receptor. Aptamers are single-stranded nucleic acid molecules that are selected from large random libraries for binding to any target molecule, much like monoclonal antibodies. In preliminary studies, we selected RNA aptamers that bind in vitro to the extracellular domain of mouse TfR. We found that such aptamers could bind in saturable fashion to cultured lymphoma cells and that a biotinylated aptamer-streptavidin conjugate could be endocytosed by cultured L cells. Specific Aim 1 is to generate modified RNA aptamers resistant to ribonuclease for subsequent studies in vivo, and to develop general procedures to conjugate aptamers to model proteins. Specific Aim 2 is to measure quantitatively the binding of the conjugates to TfR, correlating properties measured in vitro with binding and endocytosis in cell culture. Specific Aim 3 is to set up a cell culture model of the BBB for the study of transcytosis of aptamers and protein-aptamer conjugates, in order to select the most promising candidates for in vivo studies. Specific Aim 4 is to test the ability of aptamer-modified lysosomal enzymes to enter the CNS and correct the pathology in mouse models of LSD. Disease/enzymes combinations selected for study are, in order of priority, MPS I/ alpha-L-iduronidase, MPS III B/ Alpha-N-acetylglucosaminidase, MPS VII/ beta-glucuronidase and Sandhoff disease/beta-hexosaminidase. If successful, this study will provide a general approach for introducing therapeutic molecules into the brain and treating lysosomal storage disorders with CNS involvement, and may have implications for treating other neurologic diseases.

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