Recombinant LRP fragments production for Alzheimer?s disease treatment

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is associated with abnormal accumulation of amyloid-2 peptides (A2) in brain due mainly to faulty clearance. Rapid clearance from brain across the blood-brain barrier (BBB) is regulated by the low density lipoprotein receptor-related protein 1 (LRP1). LRP consists of four ligand binding domains of which domains II (LRP-II) and IV (LRP-IV) bind A240 and A242 in vitro with high affinity. Recently, we have shown that human plasma sLRP, soluble extracellular domain of LRP, is a key endogenous `brain A2 sinker'. We have also showed that in a mouse AD model (Tg 2576) treated with low levels of recombinant LRP-IV brain A2 levels are reduced, and cerebral blood flow (CBF) responses to brain activation and learning and memory are improved. Unlike other Ab `sinker' agents LRP-II and LRP-IV have the highest affinity for binding directly different A2 isoforms, and do not enter brain. Therefore, this therapeutic approach may not lead to potential neuro-inflammation as seen in immune based therapies. In our earlier studies we used purified LRP-IV produced by stably transfected Baby Hamster Kidney (BHK) cells expressing LRP-IV with a tag. For therapeutic purposes we need to produce the pure recombinant proteins, without extraneous amino acids, and to use a cell line that is acceptable to the FDA. To achieve this we propose to use Chinese hamster ovary (CHO) cells and culture the cells in a medium free of serum and proteins. The goals of Phase I are to generate an expression system for pure recombinant LRP-II and LRP-IV production, and to demonstrate that they preferentially bind A2 in vitro. Currently, there are no cures for AD, a devastating disease that affects about 4.5 million people in the US, and cost about $100 billion in patient care. The major unmet need in the AD therapeutic market is for disease modification and disease prevention drugs. Our disease modifying approach, using recombinant LRP-II or LRP-IV, which do not enter brain, may be effective in curing and preventing AD. PUBLIC HEALTH RELEVANCE Alzheimer's disease (AD) is associated with abnormal accumulation of brain toxins called amyloid-2 peptides (A2). This is due mainly to faulty clearance from brain directly into blood. Rapid clearance from brain is regulated mainly by a transporter called low density lipoprotein receptor-related protein 1 (LRP1) on the brain blood vessels. A2 directly attaches to LRP1 resulting in its clearance from brain into circulating blood. LRP consists of four regions that may bind molecules, two of which, region II (LRP-II) and IV (LRP-IV) strongly bind A2. It was shown that mice (AD model) injected with LRP-IV had lower brain A2, and this was associated with improved functional brain blood flow and learning and memory. Currently, there are no cures for AD, a devastating disease that affects about 4.5 million people in the US, and cost about $100 billion in patient care. Current drugs treat symptoms of the disease and these are of limited use in slowing the disease progression. The major unmet need in the AD therapeutic market is for disease modification and disease prevention drugs. Our disease modifying approach, using LRP-II or LRP-IV, may be effective in treating and preventing AD. This proposal will evaluate the production process of LRP-II and LRP-IV and their potential as new therapies for AD.