GB2463925A

GB2463925A - Screening for agents for the treatment of Alzheimers Disease

Info

Publication number: GB2463925A
Application number: GB0812779A
Authority: GB
Inventors: Laura Turner; Adam Kline; Joanne Taylor
Original assignee: Eisai London Research Laboratories Ltd; Eisai R&D Management Co Ltd
Current assignee: Eisai R&D Management Co Ltd
Priority date: 2008-07-11
Filing date: 2008-07-11
Publication date: 2010-04-07
Also published as: GB0812779D0

Abstract

A method of screening for an agent which reduces the activity of the proteins LCAT, PEMT or PCYT1A or increases the activity of PHOSPHO1, HMG CoA Reductase (HMGR), SP3, LYPLA2 or HNF1A (TCF1) wherein each of these proteins are enzymes involved in cholesterol metabolism. The agent may have activity in the treatment or prevention of Alzheimer's disease. The agent may cause a reduction of beta-amyloid (Aβ) levels. Genetic constructs encoding the proteins, RNAi inhibition of the expression of the proteins and a beta-amyloid specific LCAT metabolic pathway are disclosed.

Description

INTELLECTUAL

. .... PROPERTY OFFICE Application No. GBO8 12779.7 RTM Date:28 January 2010 The following terms are registered trademarks and should be read as such wherever they occur in this document: Dharmacon Intellectual Property Office is an operating name of the Patent Office www.ipo.gov.uk

I

Method of screening for agents to treat Alzheimer's Disease The present invention relates to a method of screening a candidate agent for its ability to reduce the activity of lecithin cholesterol acyl transferase (LCAT), phosphatidylethanolamine methyltransferase (PEMT) and/or phosphate cytidylyltransferase 1, choline, alpha (PCYT1A) or increase the activity of phosphatase orphan 1 (PHOSPHO 1), 3-hydroxy-3-methylglutaryl-coenzynie A reductase (HMGCR), specificity protein 3 transcription factor (SF3), lysophospholipase II (LYPLA2) andlor hepatic nuclear factor lAltranscription factor 1 (HNF1AITCFI), the method comprising screening a candidate agent and determining if the activity of LCAT, PEMT and/or PCYT1A is reduced or if the activity of PFIOSPI{O1, HMGCR, SP3, LYPLA2 and/or FINFIAITCF1 is increased.

The invention also relates to genetic constructs comprising a nucleic acid sequence encoding LCAT, PEMT, PCYTIA, PHOSPHO1, HMGCR, SF3, LYPLA2 or HNFIA/TCF1, as well as host cells which comprise such a genetic construct. The invention further relates to candidate agents identified by the screening methods of the invention and to methods of treating patients whom suffer from or whom are predisposed to suffer from Alzheimer' s Disease.

Dementia is a brain disorder that seriously affects a person's ability to carry out daily activities. The most common form of dementia among older people is Alzheimer's disease (Alzheimer's Disease), which initially involves the parts of the brain that control thought, memory, and language. There is currently no cure for Alzheimer's Disease.

Alzheimer's Disease begins slowly. At first, the only symptom may be mild forgetfulness, which can be confused with age-related memory change. In the early stage of Alzheimer's Disease, people may have trouble remembering recent events, activities, or the names of familiar people or things. They may not be able to solve simple mathematical problems.

However, as the disease goes on, symptoms are more easily noticed and become serious enough to cause people with Alzheimer's Disease or their family members to seek medical help. Forgetfulness interferes with daily activities. People in the middle stages of Alzheimer's Disease generally forget how to do simple tasks like brushing their teeth or combing their hair. They can no longer think clearly. They begin to fail to recognize familiar people and places. They have problems speaking, understanding, reading, or writing. As the disease progresses, people with Alzheimer's Disease may become anxious or aggressive, or wander away from home. Eventually, patients need total care.

It is believed that as many as 4.5 million Americans suffer from Alzheimer's Disease (and clearly more world-wide). The disease usually begins after age 60 with the risk increasing with age. While younger people may get Alzheimer's Disease, it is much less common. About 5 percent of men and women ages 65 to 74 have Alzheimer's Disease, and nearly half of those age 85 and older may have the disease. It is important to note that Alzheirner's Disease is not a normal part of aging.

Alzheimer's Disease is named after Dr. Alois Alzheimer, a German doctor. In 1906, Dr. Alzheimer noticed changes in the brain tissue of a woman who had died of an unusual mental illness. He found abnormal clumps (now called amyloid plaques) and tangled bundles of fibers (now called neurofibiillary tangles). Today, these plaques and tangles in the brain are considered signs of Alzheimer's Disease.

Scientists also have found other brain changes in people with Alzheimer's Disease.

Nerve cells die in areas of the brain that are vital to memory and other mental abilities, and connections between nerve cells are disrupted. There also are lower levels of some of the chemicals in the brain that carry messages back and forth between nerve cells. Alzheimer's Disease may impair thinking and memory by disrupting these messages.

The amyloid hypothesis postulates that Alzheimer's Disease is caused by aberrant production or clearance of the amyloid 13 (A13) peptide from the brains of affected individuals.

A13 is toxic to neurons and forms plaques in the brains of Alzheimer's Disease patients. These plaques constitute one of the hallmark pathologies of the disease.

A13 is produced by the consecutive proteolytic cleavage of the Amyloid Precursor Protein (APP) by f3-secretase (BACE) and ?-secretase proteases. APP is also cleaved by u-secretase but this process generates non-amyloidogenic products (see Figure 1).

Cleavage by y-secretase generates AJ3 peptides of variable lengths. The 42 amino acid form of A13 (A131-42) is known to be the most toxic.

There is an ongoing need in the art to identify therapeutic agents to prevent and treat Alzheimer's Disease. The present invention addresses this need.

The present invention relates to a method of screening a candidate agent for its ability to reduce the activity of lecithin cholesterol acyl transferase (LCAT), phosphatidylethanolamine methyltransferase (PEMT) and/or phosphate cytidylyltransferase I (PCYT1A) or increase the activity of phosphatase orphan! (PHOSPHO 1), 3-hydroxy-3 -methylglutaryl-coenzyme A reductase (HMGCR), specificity protein 3 transcription factor (SP3), lysophospholipase II (LYPLA2) and/or hepatic nuclear factor lA/transcription factor 1 (HNF1AiTCFI), the method comprising determining if the activity of LCAT, PEMT and/or PCYT1A is reduced or if the activity of PHOSPHO1, HMGCR, SP3, LYPLA2 and/or HNFIAITCF1 is increased, in the presence of a candidate agent.

The present invention relates to the fact that the inventors have now identified that reducing the activity of LCAT, PEMT and/or PCYT1A or increasing the activity of PHOSPHO1, HMGCR, SP3, LYPLA2 and/or HNF1A/'FCF! reduces the production of A1. These novel findings can be utilised to identify treatments for Alzheimer's Disease.

LCAT, PEMT, PCYT1A, PHOSPHO1, HMGCR, SF3, LYPLA2 and HNF1AJTCFI are proteins which have previously been reported in the literature.

LCAT, PEMT, PCYTIA, PHOSPHO1, HMGCR, SP3, LYPLA2 and HNFIAJTCF1 have never previously been reported to have any role in the regulation of AJ3 levels in Alzheimer's Disease.

LCAT is a 416 amino acid glycoprotein that is expressed in the liver, brain and testes (Warden et al. (1989) J. Biol. Chem. 264, 36, 21573). The function of LCAT is the esteiffication of cholesterol. The activity of LCAT is potentiated by ApoAl and most LCAT is associated with ApoD in high density lipoprotein (HDL) particles. LCAT deficiency causes a rare dyslipoproteinernia, resulting in corneal opacity (secondary amyloid deposits), renal failure, arteriosclerosis, anemia and proteinuria.

Enzymatically active LCAT is present in human cerebrospinal fluid (CSF) and LCAT activity has been shown to be 50% lower in the CSF of Alzheimer's Disease patients compared to normal controls (Demeester N. et a!. (2000) J. Lipid Res, Vol. 41).

These findings may have led a skilled person to consider that, in the context of Alzheimer's Disease, lowering LCAT levels may not be desirable. However, the inventors of the present invention have shown that it is in fact desirable to decrease LCAT levels, as this decreases A13 levels.

As the function of LCAT is the esterification of cholesterol, the novel finding that decreasing LCAT activity decreases A13 levels suggests that unesterified cholesterol levels should be maintained in the brain in order to treat Alzheimer's Disease. Again, this is in contrast to many previous attempts to treat Alzheimer's Disease that focused on trying to lower cholesterol in the brain.

PEMT is involved in glycerophospholipid metabolism (the same pathway that produces the LCAT substrate phosphatidyicholine). The function of PEMT is the catalysis of phosphatidyicholine synthesis. PEMT catalyses three sequential methylations of phosphatidylethanolamine (PE) by AdoMet (s-adenosylmethion me), thus producing phosphatidyicholine (PC). This gene has also been associated with hepatocyte proliferation and liver cancer. Three isoforms of PEMT exist that differ from each other in the 5' region.

PCYT1A is also involved in glycerophospholipid metabolism. The function of PCYTIA is controlling phosphatidyicholine synthesis. PCYTIA is an important enzyme for the synthesis of membrane phospholipids and it catalyses a rate limiting step in the biosynthesis of phosphatidyicholine. Two isoforms, alpha and beta exist.

LYPLA2 is another enzyme involved in glycerophospholipid metabolism. In the context of glycerophospholipid metabolism, LYPLA2 converts 2-acyl-sn-glycero-3-phosphocholi ne into sn-gl ycero-3-phosphocholine.

PHOSPHOI, a member of the haloacid dehalogenase (HAD) superfamily of Mg2+ dependent hydrolases, is a phosphoethanolamine/phosphocholine phosphatase enzyme that has been implicated in the generation of inorganic phosphate required for matrix mineralization, a process central to skeletal development (Roberts SJ et al (2004) Biochem J. 382, 59-65).

SP3 is a ubiquitously expressed zinc-finger DNA-binding domain transcription factor that can act as an activator or repressor of transcription in a context dependent manner.

HNF1A/TCFI is a member of the T-cell factor (Tcf) family of activators/repressors that are important for many developmental processes. Tcf factors are transcriptionally inert, but they become potent transactivators following interaction with the Wnt signalling protein 3-catenin. HNF1 is involved in the regulation of many hepatic genes.

HMGCR is a transmembrane glycoprotein that is involved in the control of cholesterol biosynthesis. It is the rate limiting enzyme of sterol biosynthesis.

LCAT, PEMT, PCYT1A, PHOSPHO1, HMGCR, SP3, LYPLA2 and HNF1A/TCF1 in accordance with the invention means the LCAT, PEMT, PCYT1A, PHOSPFIOI, HMGCR, SP3, LYPLA2 and HINFIA/TCFI protein respectively.

LCAT, PEMT, PCYT1A, PHOSPHO!, I-IMGCR, SP3, LYPLA2 and HNF1A/TCFI activity according to the invention means the level of expression of each gene encoding LCAT, PEMT, PCYT1A, PHOSPHO1, HMGCR, SP3, LYPLA2 and HNFIAJTCF1 respectively, the amount of LCAT, PEMT, PCYT1A, PHOSPHOI, HMGCR, SP3, LYPLA2 and HNF1A/TCF1, or the rate at which LCAT, PEMT, PCYT1A, PHOSPHOI, HMGCR, SP3, LYPLA2 and HNF1A/TCFI perform their respective functions.

The method of the invention comprises contacting LCAT, PEMT, PCYTIA, PHOSPHOI, HMGCR, SP3, LYPLA2 and/or HNF1AJTCFI with a candidate agent in order to determine if the candidate agent reduces LCAT, PIEMT and/or PCYTIA activity or increases PHOSPHO1, I-IMGCR, SP3, LYPLA2 and/or HNF1A/TCFI activity.

The method of the invention can comprise contacting a host cell which expresses LCAT, PEMT, PCYT1A, PHOSPHOI, HMGCR, SP3, LYPLA2 and/or HNF1A/TCF1 with a candidate agent. The method of the invention can also comprise contacting LCAT, PEMT, PCYTIA, PHOSPHOI, HMGCR, SP3, LYPLA2 and/or HNF1A/TCFI in culture with a candidate agent or isolated LCAT, PEMT, PCYTIA, PFIOSPHO1, HMGCR, SP3, LYPLA2 and/or HNF1AICF1 with a candidate agent.

The LCAT, PEMT, PCYT1A, PHOSPHO!, HMGCR, SP3, LYPLA2 and/or HNF1A/TCF1 of the invention can be natural or recombinant.

Determination of whether LCAT, PEMT and/or PCYT1A activity is reduced or whether PHOSPHOI, HMGCR, SP3, LYPLA2 and/or HNF1A/TCFI activity is increased can be by any method known in the art. For example, the amount of LCAT, PEMT, PCYT1A, PHOSPHO1, HMGCR, SP3, LYPLA2 and/or HNF1AJTCF1 can be determined using various antibody-based detection strategies. These include Western blotting, Enzyme linked immunosorbent assay (ELISA) or Homogeneous Time-Resolved Fluorescence (HTRF). The reduction/enhancement of the rate at which LCAT, PEMT, PCYT1A, PHOSPHO1, HMGCR, SP3, LYPLA2 and/or FINF1A/TCFI perform their respective functions can be determined by any method and can include monitoring changes using methods such as competitive immunoassay with fluorophore-tagged antibodies or reporter assays.

In one aspect, the method comprises contacting LCAT, PEMT, PCYTIA, PHOSPHO1, HMGCR, SP3, LYPLA2 or HNF1A/TCFI substrate with LCAT, PEMT, PCYT1A, PHOSPHOI, HMGCR, SP3, LYPLA2 or HNF1A/TCF1 respectively in the presence of a candidate agent.

A substrate in accordance with the invention is any reactant consumed as LCAT, PEMT, PCYT1A, PHOSPHOI, HMGCR, SP3, LYPLA2 or HNF1A/TCF1 performs its function.

A LCAT substrate includes any source of phosphatidylcholine or cholesterol (LCAT esterifies cholesterol and some other steroids/sterols at the 3-1 hydroxyl e.g. pregnenolone, dehydroepiandrosterone, estradiol). Artificial proteoliposome substrates, composed of egg yolk lecithin, apo Al purified from human plasma and unesterified cholesterol (IJC), can also be used. LCAT substrates include artificially synthesised substrates or plasma.

LCAT enzyme activity can be measured using a simple fluorimetnc assay that aims to measure the cleavage of phosphatidyicholine by LCAT. An artificial LCAT substrate is provided that fluoresces at 470nm in its uncleaved state. Following cleavage by LCAT, a monomer is released that fluoresces at 39Orim. The ratio of 470/390 should decrease with time following LCAT addition. If compounds that inhibit LCAT activity are added to this assay, the decrease in the 470/390 ratio should no longer occur across time.

LCAT enzyme activity can also be measured by assessing the rate of cholesterol esterification, for example in plasma. In this case, LCAT activity is expressed as the molar esterification rate (MER), which represents the quantity of unesterified cholesterol (UC) esterified by the source of LCAT in units of time. In these experiments, LCAT activity is usually measured by counting the amount of UC labelled with 3H or 4C that has been incorporated into cholesterol esters.

Examples of PEMT substrates are S-adenosyl-L-methionine and phosphatidylethanolamine.

PEMT activity can be determined by measuring the incorporation of [3H]-methyl groups from S-adenosyl-l-(methyl-3H)-methionine (SAM) into phospholipids, e.g. from intestinal brush border membranes (BBM) or microsomal phosphatidylethanolamine (PE). This can be monitored by scintillation counting.

Specific activity of the enzyme is expressed as femtomoles of [31-1]-methyl groups incorporated into phospholipids/mg of protein/incubation time at 37°C (Castano JG et al.(1980) J. Biol. Chem. 255, 90419043).

Examples of PCYT1A substrates are cytidine triphosphate (CTP) and phosphocholine/choline phosphate.

Changes in PCYT1A activity can be measured by monitoring changes in the phosphorylation status of PCYT1A, for example by using phospho-specific antibodies directed to Serine 315, or by following a shift in the molecular weight of PCYTIA by western analysis. Phosphorylation of PCYT1A at Senne 315 has been shown to decrease its activity, and thus decrease synthesis of phosphatidyicholine. The phosphorylation of PCYTIA can be stimulated using the oxysterol 22-hydroxycholesterol (22-HC) in combination with its obligate partner, 9-cis-retinoic acid (9-cis-RA), which leads to a decrease in phosphatidyicholine synthesis. The effects of 22-HC:9-cis-RA are dependent upon signalling through ERK p42 (Agassandian M et a! (2005) JBC 280, 2 1577-87). Phospho-specific antibodies can also be used in an HTRF assay performed to follow PCYT1A activity. PCYT1A activity can also be monitored using [4C] methyl phosphoryicholine and CTP as substrates in a PCYTIA activity assay and measuring the incorporation of [4C] methyl phosphoryicholine into CDP-phosphocholine, as analysed by thin-layer chromatography and scintillation counting. For these assays, PCYT1A can be obtained from hippocampal neuron cultures produced from E17 or E18 Wistar rat or mouse brains. As a positive control for this assay, active site-directed inhibitors of glucocerebrosidase will elevate PCYTIA activity (Bodennec J et a! (2002) Faseb J. 16, 1814-18 16).

Examples of PHOSPHOI substrates are 0-phosphoethanolamine and phosphocholine.

PHOSPHO1 activity can be measured by using a phosphatase assay to monitor the dephosphorylation of 0-phosphoethanolamine or phosphocholine by PHOSPHO1 to liberate ethanolamine or choline plus phosphate. Phosphatase assays for PHOSPHO1 activity can be performed as described by Roberts SJ et a! (2004) Biochem. J. 382, 59-65. Briefly, recombinant PHOSPHO1 is produced by expressing a cDNA construct encoding PHOSPHOI fused to a VS epitope and 6 His-tag at the C-terminus in E. coli. Pure recombinant PHOSPHOI can then be isolated using a Ni-NTA-agarose column, and dialysed in TBS, pH 7.2 to produce a stock for use in subsequent assays.

Western analysis can confirm the purity of the PHOSPHO1 protein. Standard discontinuous colorimetric assays can be performed, using purified recombinant PHOSPHOI in the presence of the corresponding divalent metal chloride salt and a substrate (BayKov AA et a! (1988) Anal. Biochem. 171, 266-270). Standard solutions containing known concentrations of KH2PO4 can be included as controls. Reactions proceed for 15 mm at 37°C, and then stopped by the addition of sulphuric acid containing ammonium molybdate, Tween 20 and Malachite Green. The absorbance of each well at 630 nm is measured and the specific activity is calculated in units of activity per mg of enzyme, where I unit of activity represents the hydrolysis of 1 nmol of phosphate per mm. Alternatively, a continuous spectrophotometric assay can be performed using the EnzChek� Phosphatase Assay Kit (Molecular Probes, Eugene, OR, U.S.A.), which is based upon the punne nucleoside phosphorylase (PNPase)-coupled assay.

Examples of HMGCR substrates are (S)-3-hydroxy-3-methylglutaryl-CoA and 2 NADPH.

HMGCR activity can be measured by monitoring the production of mevalonate, CoA and 2 NADP(+) from (S)-3-hydroxy-3-methylglutaryl-CoA and 2 NADPFI according to a standard method by Brown eta! (1979) J. Biol. Chem. 254, 5144-5 149. FIIvIGCR activity can be assayed from many cells types (e.g. human fibroblasts, liver cells) or tissues (e.g. rat liver) or microsome preparations (small vesicles derived from smooth endoplasmic reticulum following homogenisation) isolated from liver tissue, by measuring the rate of formation of [S-'4C] mevalonate from [314C1 hydroxymethylglutaryl-CoA. Cell/tissue extracts containing HMGCR are incubated at 37°C in a buffer containing K2HPO4, (pH 7.5); glucose-6-phosphate TPN; glucose-6- phosphate dehydrogenase and dithiothreitol. The reaction is started by addition of DL- [3'4C]hydroxymethylglutaryl CoA. After 120 mm at 37°C the reaction is stopped by addition of 5 N HCI; [3H]mevalonolactone is added as an internal standard, and the mixture is extracted with diethyl ether. The mevalonolactone is isolated by thin-layer chromatography and analysed for radioactivity, which is quantified by scintillation counting. It may also be possible to use LDL-receptor levels in liver cells as a readout for HMGCR activity, as inhibition of HIMGCR in the liver stimulates the LDL-receptors, which results in an increased clearance of LDL from the bloodstream and a decrease in blood cholesterol levels. In addition, hepatic HtvIGCR exists in a phosphorylated (inactive) and dephosphorylated (active) form. It may be possible to monitor the activation status of I-IIvIGCR using a phospho-specific antibody raised against an inactive form of I-IMGCR. This could then be used in standard FITRF or western blotting assays. In this case, E. co!i alkaline phosphatase can be used to maximally activate the enzyme by removing a phosphate group. Enzyme activity after phosphatase treatment may be used as a measure of total HMGCR activity.

An example of a SP3 substrate is a promoter element of any gene that contains a GT or GC box motif and an example of a HNFIA/TCFI substrate is any nucleotide sequence contai fling the inverted palindrome 5' G11'AATNATT'AAC-3'.

HNF1AITCFI and SP3 are both transcription factors. Reporter constructs can be used to monitor the activity of these transcription factors in cell based assays, where the reporter constructs will be expressed, in the presence or absence of HNFIA/TCFI or SP3, and the activity of endogenous HNF1A/TCFI or SP3 analysed. These constructs can consist of a reporter gene, for example luciferase or f3-galactosidase, tagged at the 3' end of a promoter sequence from any gene known to contain a binding site for HNF1AITCF1 or SP3. Binding of the transcription factors to their target sites within the promoters would lead to the initiation of transcription of the reporter gene by RNA polymerase. The reporter gene would thus provide a readout of HNFIA/TCF1 or SP3 activity within the cell (either by performing a luciferase assay (Promega), or assaying for -galactosidase activity. In the example of HNF1AITCF1, Wnt3a is known to significantly increase the activity of this transcription factor, so this can be used as a positive control for transcriptional upregulation. In cases where these transcription factors are acting in an inhibitory manner, binding of the transcription factor to the reporter construct would lead to an inhibition of constitutive transcription from these constructs.

Examples of LYPLA2 substrates are a S-acylated cysteine residue in proteins such as tnmeric 0 alpha protein or FIRAS and any palmitoylated protein. In the context of gI ycerophospholipid metabolism, a LYPLA2 substrate includes 2-acyl-sn-glycero-3-phosphocholine.

Lysophospholipase activity of LYPLA2 activity can be assayed by measuring the hydrolysis of [4C] palmitic acid from l-['4C]palmitoyllyso-GPC. In order to do this organic solvents are evaporated from the lysophospholipid substrate followed by the addition of 50 mM Tris, pH 9, and then sonication. The lysophospholipase reaction contains; l-[14C]palmitoyllyso-GPC, CaCI,, and human serum albumin in 50 mM Tris, pH 9. The reaction is stopped by addition of Dole reagent, which was followed by the addition of heptane and water. Unlabeled paimitic acid is also added as a cold carrier.

After vortexing, the upper phase is removed and evaporated, and the amount of radioactivity is determined by liquid scintillation spectrometry (Leslie CC (1991) J. Biol. Chem. 266, 11366-11371).

When the method of the invention comprises contacting a host cell which expresses LCAT, FEMT, PCYT1A, PHOSPHO1, HMGCR, SF3, LYPLA2 and/or HNF1A/TCF1 with a candidate agent, the host cell can be any cell which expresses LCAT, PEMT, PCYTIA, PHOSPHOI, HIMGCR, SF3, LYPLA2 and/or HNFIA/TCF1. The host cell may be genetically engineered to contain exogenous DNA encoding LCAT, PEMT, PCYT1A, PHOSPHO1, HMGCR, SP3, LYPLA2 and/or 1-INF1A/TCFI or engineered to increase the expression of LCAT, PEMT, PCYT1A, PFIOSPHOI, HMGCR, SF3, LYPLA2 and/or HINFIA/TCFI (exogenous or endogenous) or not genetically altered if the cell already expresses LCAT, PEMT, PCYT1A, P}IOSPHO1, HMGCR, SP3, LYPLA2 and/or HNF1A/TCF1 A suitable host cell can be any cell including a prokaryotic or eukaryotic cell. In particular, the host cell can be a yeast cell, baculovirus infected insect cell, mammalian cell line or human cell lines (which can be immortilised), including human embryonic ce]l kidney cells, such as HEK293, as well as CHO or COS cells.

Determination of whether LCAT, PEMT, PCYT1A, PHOSPHO1, HMGCR, SP3, LYPLA2 and/or HNFIA/TCF1 activity is reduced/enhanced can be by any method known in the art as discussed above. In addition, such methods can include contacting the host cells with a candidate agent and determining the amount of LCAT, PEMT, PCYTIA, PHOSPHO!, HMGCR, SP3, LYPLA2 and/or HNF1AiTCFI, compared to the amount of LCAT, PEMT, PCYTIA, PHOSPHO!, i-IMGCR, SF3, LYPLA2 and/or HNF1AJTCF1 expressed in the absence of a candidate agent.

Alternatively, a comparison can be made by introducing, into the host cell, a nucleic acid which encodes a reporter gene and which is expressed alongside LCAT, PEMT, PCYTIA, PHOSPHOI, I-IMGCR, SP3, LYPLA2 or HNFIA/TCF1. In such a situation, the level of the reporter gene directly correlates to the level of LCAT, PEMT, PCYT1A, PHOSPHO1, HMGCR, SP3, LYPLA2 or HNFIA/TCF1 gene which has been expressed. In this situation, the reporter gene is usually produced as a fusion protein together with the expression of LCAT, PEMT, PCYT1A, PHOSPHO1, I-IIvIGCR, SP3, LYPLA2 or FINF1AITCFI. Where the host cell also expresses (produces) A3, the reduction of activity of LCAT, PEMT or PCYT1A, or the increase in activity of PHOSPHOI, HMGCR, SP3, LYPLA2 or HNFIAITCF1, can also be determined by monitoring changes in A(3 levels.

The present invention particularly relates to the identification of agents which can effect and reduce the activity of LCAT, PEMT and/or PCYTIA, or increase the activity of PHOSPHO1, H1MGCR, SP3, LYPLA2 and/or HNFIAITCFL, and therefore cause a reduction in AJ3 production. Reduction in A3 production will reduce the occurrence of and the problems associated with Alzheimer's Disease. Accordingly, identifying agents which reduce the activity of LCAT, PEMT and/or PCYT1A, or increase the activity of PHOSPHO1, HMGCR, SP3, LYPLA2 and/or HNF1A/TCF!, is extremely useful for those patients whom suffer from or whom are predisposed to suffer from Alzheimer' s Disease.

In accordance with the invention, candidate agents can reduce the activity of LCAT, PEMT and/or PCYTIA, or increase the activity of PHOSPHO1, HMGCR, SP3, LYPLA2 and/or HNFIAJTCFI, by any means (including after production of the protein and also reducing production of the protein). Preferably, the agent acts by directly reducing the activity of the expressed LCAT, PEMT and/or PCYTIA protein (i.e. it is a protein inhibitor) or directly increasing the activity of the expressed PHOSPHOI, HMGCR, SP3, LYPLA2 and/or HNFIAII'CFl protein. Alternatively, the agent can act by reducing the LCAT, PEMT and/or PCYT1A gene expression levels (which clearly also reduces the amount of LCAT, PEMT and/or PCYTIA and the rate at which LCAT, PEMT and/or PCYTIA perform their respective functions), or increasing the PHOSPHOI, HMGCR, SP3, LYPLA2 and/or HNFJAITCFI gene expression levels (which clearly also increases the amount of PHOSPHO1, 1-IMGCR, SP3, LYPLA2 and/or HNF1AITCF1 and the rate at which PHOSPHO1, HMGCR, SP3, LYPLA2 and/or HNFIAITCF1 perform their respective functions). Candidate agents can be potential RNAi/antisense gene-therapy therapeutics. Reducing the activity of LCAT, PEMT and/or PCYT1A, or increasing the activity of PHOSPHOI, HMGCR, SP3, LYPLA2 and/or HNFIAJTCF1 can be achieved by the candidate agent interfering with the activity of LCAT, PEMT, PCYT1A, PHOSPHO!, HIvIGCR, SP3, LYPLA2 and/or H1NFIA/TCF1 binding partners either upstream or downstream in order to reduce Al production. Alternatively, the candidate agent may reduce the activity of LCAT, PEMT and/or PCYT1A, or increase the activity of PHOSPFIO1, HMGCR, SP3, LYPLA2 and/or HNF1A/TCFI, by effecting the nucleic acid encoding LCAT, PEMT, PCYT1A, PHOSPHO1, HMGCR, SP3, LYPLA2 and/or HNFIA/TCF1 or by effecting the expression of the protein or by effecting the maintenance or activity of LCAT, PEMT, PCYT1A, PHOSPHOI, HIMGCR, SP3, LYPLA2 and/or HNF1AITCF1 protein once produced, all of which reduce the production of A13.

Suitable screens for the present invention are those commonly known in the art and are as discussed in Gupta A, Devi LA. et al., Strategies for Screening and Drug Development, AAPS Journal 2006 8(1): E153-E159 and Coward P, Conklin BR.

Anal Biochem. 1999 270(2): 242-8.

The candidate agent in accordance with the invention can be any natural or synthetic molecule, for example small chemical molecules, proteins or nucleic acids (including siRNA molecules). The candidate agent can be molecules which are known or they can be novel molecules.

In accordance with the present invention, reference to LCAT, PEMT, PCYT1A, PHOSPHOI, HMGCR, SP3, LYPLA2 or HNFIAITCFI is to the LCAT, PEMT, PCYT1A, PHOSPFIO1, HMGCR, SP3, LYPLA2 or HNF1AITCFI amino acid sequence respectively given herein in sequence 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or a sequence which is at least 80% identical over a length of at least 15, at least 20 or at least 30 amino acids. Preferably, the sequence is at least 90% identical over the lengths specified. Preferably the sequence is 95% identical over the lengths specified.

Sequence similarity can be determined using the BLAST alignment program publicly available at http://www.ncbi.nlm.nih.gov/BLAST/.

A second aspect of the invention relates to a genetic construct comprising nucleic acid encoding LCAT, PEMT, PCYT1A, PHOSPHO1, HMGCR, SP3, LYPLA2 or 1-INF1AJTCF1 under the control of a constitutive or inducible promoter, suitable for expression of LCAT, PEMT, PCYTIA, PHOSPHO1, HMGCR, SP3, LYPLA2 or FINIFIAITCF1. This genetic construct is typically called an expression vector. Such a genetic construct can be used in accordance with a third aspect of the invention which provides a host cell comprising the construct. The genetic construct of the second aspect and the host cell of the third aspect are suitable according to the first aspect of method of screening a candidate agent according to the invention.

Optionally, the host cell of the third aspect of the invention may express (produce) Aft A fourth aspect of the invention relates to an agent that reduces the activity of LCAT, PEMT, PCYT1A, PHOSPHO1, HMGCR, SP3, LYPLA2 and/or HNFIAITCF1, which is identified by the method of the first aspect of the invention.

Such an agent includes any nucleic acid which is a short interfering nucleic acid (RNA or DNA or other nucleic acid) which is preferably 21, 22 or 23 bases in length, double stranded and has a strand complementary to nucleic acid which encodes a part of the LCAT, PEMT, PCYTIA, PHOSPHO1, HMGCR, SP3, LYPLA2 or HNF1A1TCFI amino acid sequence referred to herein. Particular short interfering molecules include those described as anti-LCAT siRNA#1, anti-LCAT siRNA#2, anti-PEMT siRNA# 1, anti-PEMT siRNA#3, anti -PCYT 1 A siRNA#5, anti -PCYT 1 A siRNA#6, anti -PHOSPHO 1 siRNA# 1, anti -PHOSPHO 1 siRNA#2, anti -PHOSPHO I siRNA#3, anti-HIIVIGCR siRNA#1, anti-HMGCR siRNA#3, anti-SP3 siRNA#1, anti-SP3 siRNA#5, anti-SP3 siRNA#8, anti-LYPLA2 siRNA#8, anti-LYPLA2 siRNA#9, anti-LYPLA2 siRNA#10, anti-HNF1A/TCF1 siRNA#1, anti-HNFIA/TCFL sIRNA#2 and anti-HNFIAITCF1 siRNA#4 in the examples section below and in table 1. Other agents identified include small chemical moieties, proteins and other nucleic acids.

A fifth aspect of the invention relates to a method of treating a patient who suffers from or whom is predisposed to suffer from Alzheimer's Disease, the method comprising screening a candidate agent according to the first aspect of the invention, identifying an agent that decreases the activity of LCAT, PEMT and/or PCYTIA, or increases the activity of PHOSPHO1, HMGCR, SP3, LYPLA2 and/or HNFIA/TCFI, and administering the agent to the patient. Preferably, the patient is in need of treatment for Alzheimer's Disease. A sixth aspect of the invention relates to a method of treating a patient who suffers from or whom is predisposed to suffer from Alzheimer's Disease by administration of a therapeutic agent, wherein the therapeutic agent is an agent which reduces the activity of LCAT, PEMT and/or PCYT1A, or increases the activity of PHOSPHO1, HMGCR, SP3, LYPLA2 andJorHNF1AJTCFI.

Such an agent may have been obtained from a method according to the first aspect of the invention. Again, preferably the patient is in need of treatment. Treatment can be therapeutic or prophylactic.

A seventh aspect of the invention provides an agent, which reduces the activity of LCAT, PEMT and/or PCYT1A, or increases the activity of PHOSPHOI, HMGCR, SP3, LYPLA2 and/or HNFIAJTCF1, for use in treating Alzheimer's Disease. An eighth aspect provides the use of an agent which reduces the activity of LCAT, PEMT and/or PCYTIA, or increases the activity of PHOSPHO1, HItvIGCR, SP3, LYPLA2 and/or HNT1AITCFI, in the manufacture of a medicament for treating Alzheimer's Disease. A ninth aspect of the invention provides a pharmaceutical composition comprising an agent which reduces the activity of LCAT, PEMT and/or PCYTIA, or increasing the activity of PHOSPHO1, FIMGCR, SP3, LYPLA2 and/or HNFIA/TCFI Such an agent can be any as described according to the fourth aspect of the invention in combination with a pharmaceutically acceptable excipient. Such a combination can be referred to as a pharmaceutical composition. Such a pharmaceutical composition can be used to treat Alzheimer's Disease.

The invention also provides a method of screening an agent which reduces the activity of LCAT, PEMT and/or PCYT1A, or increases the activity of PHOSPHO1, HMGCR, SP3, LYPLA2 and/or HNF1AITCFI, for its ability to decrease A production, the method comprising contacting a cell which expresses A with the agent and determining its ability to decrease Af3 production. Screens to determine the levels of Aj3 from a cell are known in the art. The host cell may have been engineered such that it has an increased level of A production. The APP gene of such a cell, from which A13 is produced may be endogenous or exogenous and may contain mutations known to increase A3 production such as the Swedish APP67O/671 familial Alzheimer's Disease mutation. A suitable cell line is the cell line deposited on 3 January 2007 at the ECACC under depositor reference ELL1N and ECACC accession number 07010301. In this aspect of the invention, the screen to determine the ability of the agent to decrease Aj production is a screen to determine the level (or degree) to which A13 production is decreased. This screen is particularly useful to determine the effectiveness of the agent at reducing A production.

All preferred embodiments of the first to ninth aspects of the invention, also apply to each other aspect, mutatis inutandis.

The present invention is described with reference to the following figures and tables, in which: Figure 1 illustrates processing of the Amyloid Precursor Protein (APP) by 3, a and y-secretase. Cleavage of APP by f3-secretase (BACE) generates soluble APPf3 (sAPPf) and a membrane-bound C-terminal fragment, f3CTF. The 3CTF is cleaved by y-secretase to generate A and the APP intracellular domain (AICD) fragment.

Cleavage of APP by u-secretase generates soluble APPu (sAPPa) and a membrane-bound C-terminal fragment, aCTF. The aCTF is cleaved by y-secretase to generate the non-amyloidogenic peptide P3 and the APP intracellular domain (AICD) fragment; Figure 2 illustrates sequence 1: LCAT amino acid sequence: Protein ID: NP_000220 from nucleotide REFSEQ: accession NM_000229; Figure 3a illustrates sequence 2: PEMT isoforni 1 amino acid sequence: Protein ID: NP_680477 from nucleotide REFSEQ: accession NM_148 172 and Figure 3b illustrates sequence 3: PEMT isoform 2 amino acid sequence: Protein ID: NPJ)09100 from nucleotide REFSEQ: accession NM_007 169; Figure 4 illustrates sequence 4: PCYTIA amino acid sequence: Protein ID: NP_005008 from nucleotide REFSEQ: accession NM_005017; Figure 5 illustrates sequence 5: PHOSPHO! amino acid sequence: Protein ID: NP_848595 from nucleotide REFSEQ: accession NM 178500; Figure 6 illustrates sequence 6: HMGCR amino acid sequence: Protein ID: NP_000850 from nucleotide REFSEQ: accession NM_000859; Figure 7a illustrates sequence 7: SP3 isoform I amino acid sequence: Protein ID: NP_003102 from nucleotide REFSEQ: accession NM_003111 and Figure 7b illustrates sequence 8: SP3 isoform 2 amino acid sequence: Protein ID: NP_001017371 from nucleotide REFSEQ: accession NM_00 1017371; Figure 8 illustrates sequence 9: LYPLA2 amino acid sequence: Protein ID: NP...

00919 1 from nucleotide REFSEQ: accession NM_007260 Figure 9 illustrates sequence 10: HNF1A/TCF1 amino acid sequence: Protein ID: NP_000536 from nucleotide REFSEQ: accession NM_000545 Figure 10 illustrates inhibition of endogenous A131-40 production in human neuroblastoma cells by LCAT knockdown (validation of LCAT as a target). Anti-LCAT siRNAs decreased production of A31-40 in human neuroblastoma cells. A non-targeting siRNA was used as a negative control (NTC) and a siRNA targeting a gene known to be involved in A13 production was included as a positive control (Pos cont). None of the siRNAs used substantially reduced cell viability as determined by Alamar Blue assay; Figure ha illustrates an siRNA resistant LCAT mutant (LCATmut#1#2-V5) rescuing the ability of anti-LCAT siRNA #1 and #2 to decrease A131-40 production (Experiment 1 of rescue validation of LCAT as a target). Figure lIb is a Western Blot that illustrates that LCATmut#l#2 is resistant to LCAT siRNAs #1 and #2; Figure 12a illustrates an siRNA resistant LCAT mutant (LCATmut#1#2-V5) rescuing the ability of anti-LCAT siRNA #1 and #2 to decrease Af31-40 production (Experiment 2 of rescue validation of LCAT as a target). Figure 12b is a Western Blot that illustrates that LCATmut#1#2 is resistant to LCAT siRNAs #1 and #2; Figure 13 illustrates inhibition of endogenous A1-40 production in human neuroblastoma using DTNB, a pharmacological inhibitor of LCAT activity. The "%dF" value is representative of Afll-40 levels; Figure 14 illustrates inhibition of endogenous A131-40 production in human neuroblastoma using PCMPS, a pharmacological inhibitor of LCAT activity; Figure iSa illustrates a schematic glycerophospholipid diagram of the potential role that PEMT, PCYTIA, PHOSPHOI, LYPLA2 play in the LCAT pathway. Figure lSb illustrates a schematic cholesterol metabolism diagram of the potential role that HMGCR plays in the LCAT pathway; Figure 16a illustrates an increase in endogenous A31-40 production in human neuroblastoma cells by LYPLA2 knockdown. Figure 16b is a Western Blot illustrating that LYPLA2 does not consistently (?) appear to alter APP (FL-APP and CTF) levels; Figure 17a illustrates an increase in endogenous A1-4O production in human neuroblastoma cells by H1NF1A/TCFI knockdown. Figure 17b illustrates an increase in endogenous A31-40 production in human neuroblastoma cells by PHOSPHOI knockdown. Figure 17c is a Western Blot illustrating that siRNAs against both HNF1A and PHOSPHO1 increase full length APP (FL-APP) and APPc-terminal fragment (CTF) levels; Figure 18a illustrates inhibition of endogenous A131-40 production in human neuroblastoma cells by PEMT knockdown. Figure 18b illustrates an increase in endogenous AJ31-40 production in human neuroblastoma cells by HMGCR knockdown. Figure 18c illustrates an increase in endogenous A1-4O production in human neuroblastoma cells by SP3 knockdown. Figure 18d is a Western Blot illustrating that PEMT does not appear to alter APP (FL-APP and CTF) levels and that HMGCR and the two significantly efficacious anti-SP3 siRNAs increase full length APP (FL-APP) and APPc-terminal fragment (CTF) levels and Figure 19a illustrates inhibition of endogenous A131-40 production in human neuroblastoma cells by PCYTIA knockdown. Figure 19b is a Western Blot illustrating that PCYT1A does not appear to alter full length APP (FL-APP) levels.

In all graphs illustrated in the above figures, an asterisk placed over a pair of bars i.e. samples indicates that these are the samples that all other data was expressed as a % of; in the case where two pairs of bars have an asterisk over them in the same graph, a dashed vertical line separates the graph into two parts and the asterisks indicate the samples that all other data was expressed as a % of within that part of the graph.

TabLe 1 describes the siRNAs used in LCAT pathway analysis and Table 2 provides a summary of results in relation to genes identified from the LCAT pathway analysis.

The invention will now be described by way of reference to the following Examples, which are provided for the purposes of illustration only and are not to be construed as being limiting to the invention.

Examples

RNA interference (RNAi) is a recently discovered functional tool. This is a phenomenon where an RNA introduced into a cell ultimately causes the degradation of the complementary cellular mRNA and leads to the knockdown of gene activity.

RNAi is exploited experimentally by transfecting cultured cells with short interfering RNAs (siRNAs). These are short 21-23 nucleotide RNA duplexes complementary to the target mRNA.

Experimental details ELL1Ngrowth conditions Human neuroblastoma cells used in some of the experiments are a cell line having a constitutive high level of At31-40 production, which were deposited at The European Collection of Cell Cultures (ECACC), on 3 January 2007, under the ECACC accession number 07010301 and depositor reference ELL1N.

11EK293 cells were also used in some expenments.

Cells were cultured in a humidified atmosphere of 10% CO2 at 37°C in a 1:1 mix of Minimum Essential Medium (Sigma-Aldrich #M2279) and HAM'S F12 medium (Invitrogen #21765-029) supplemented with 15% (v/v) bovine foetal calf serum (PAA laboratories #A15-003), a 1:100 dilution of 100X Non-Essential Amino Acids (Sigma-Aldrich #M7145), 100 units/mI penicillin and 100.tg/ml streptomycin (Invitrogen #15140-114).

sIRNA screeninj protocol ELLIN cell stocks were trypsinised (Invitrogen #25300-054) and replated 1:2 the day before transfection. On the day of transfection cells were again trypsinised and subsequently replated in serum and antibiotics free media at 3*75* i� cells per 8Oiil media per well of a 96-well plate.

Transfection reaction complexes were prepared iii Optimem (Invitrogen #51985-026) using either 1tl of Oligofectamine (Invitrogen #11668-027) or 0.2p1 Dharmafect 4 (Dharmacon # T-2004-02) per well of cells to be transfected. Under standard conditions sRNAs were screened at lOOnM final concentration on the cells.

Plated cells were incubated with transfection reactions for 4 hours. The serum free media on the cells was then made up to a final concentration of 5% (vlv) bovine foetal calf serum, 100 units/mI penicillin and 100 pg/ml streptomycin.

72 hours after siRNA transfection, cell media were harvested for analysis of A3 levels. Af31-40 levels were determined using an ELISA kit purchased from Biosource (#KHB3482) or an ELISA kit purchased from WAKO Chemicals Gmbh (#294- 62501). AISII-40 HTRF assays were carried out using a kit purchased from CisBIO (#62B4OPEB). Cell viability was determined using Alamar Blue (Biosource #DAL1O25).

Cell lysates were also harvested and both full-length APP and APP C-terminal fragment levels analysed by Western blotting using an anti-APP antibody (1:2000 Invitrogen #51-2700).

A negative control of randomised, non-targeting siRNA and a positive control s1RNA were included.

Anti-LCAT siRNAs: Annealed anti-LCAT siRNA oligonucleotides were purchased from Dharmacon (#MU-009623-0O-0002).

siRNA#1 Sense oligo: 5' -GUAGACUGCUGGAUCGAUAUU-3' Antisense oligo: 5' -P-UAUCGAUCCAGCAGUCUACUTJ-3' siRNA#2 Sense oligo: 5'-UGCAGAACCUGGUCAACAAUU-3' Anti sense oligo: 5' -P-UUGUEJGACCAGGUTJCUGCAUU-3' siRNA#3 Sense oligo: 5'-GAAAGAGGAGCAGCGCAUAUU-3' Antisense oligo: 5'-P-UAUGCGCUGCUCCUCUUUCUU-3' siRNA#4 Sense oligo: 5' -GAUACAGCAUCUCAACAUGUU-3' Antisense oligo: 5'-P-CAUGU1JGAGAUGCUGUAUCUU-3' Example 1 Primary screen A primary screen was conducted using a focused library of Dharmacon siRNA smartpools targeting 2534 genes (smartpools are mixed pools of four siRNAs targeting the same gene). Endogenous Aj1-40 production in the human neuroblastoma cell line ELL1N was measured.

The results from the primary screen illustrated that the siRNA smartpool that targeted the LCAT gene reduced endogenous A13 production in human neuroblastoma cells.

Example 2 Validation -Deconvolution of the ori2inal smartpools Each of the four LCAT siRNAs from the LCAT smartpool were tested individually to confirm that at least 2 out of 4 decreased A1-4O levels in ELLIN cells. siRNAs were transfected into ELL1N cells as discussed above under the siRNA screening protocol. After 72 hours, cell viability was assessed using Alamar Blue and A11-40 levels were determined using an HTRF kit (CisBIO). A non-targeting siRNA (NTC) was used as a negative control and a siRNA targeting a gene that encodes a protein known to be involved in A3l-40 production was used as a positive control.

Figure 10 illustrates that anti-LCAT siRNAs #1 and #2 decrease A131-40 levels.

Example 3 Validation -Rescue experiments Tertiary validation was preformed by testing whether it was possible to rescue LCAT by expressing a form of LCAT refractory to siRNA. This validation step demonstrates if the effects on A3 levels are specific to the loss of LCAT activity from the cells.

A V5-tagged, siRNA resistant LCAT eDNA (LCATmut#1#2-V5) was generated by site-directed mutagenesis from a wild-type V5-tagged cDNA clone. The sequence of LCAT primers used to generate LCATmut#1#2-V5 are shown below: bases underlined are those changed from the original LCAT sequence (NM000229), used to generate the siRNA resistant mutant. The binding sites for the corresponding Dharmacon siRNA oligos are in italics.

LCATmut#1 5'-cc ctt ggg gui ga tg igg at gat ac ace agg gtt g -3' LCATmut#2 5' ac aca ctg gtg cag aaL ct gtg aac aat ggc tac gtg c -3' Mutating three bases within the target sequences of the anti-LCAT siRNA's #1 and #2 is sufficient to prevent these siRNAs from decreasing mRNA levels of this cDNA when overexpressed. This does not change the amino acid sequence of LCAT, as the mutations are all silent mutations in the third base of the codon.

HEK293 cells were transfected with the indicated siRNAs. After 24hrs, the cells were transfected again with the indicated cDNAs (pcDNA3. 1 was used as an empty vector control). All cDNA transfections were carried out using the Lipofectamine 2000 reagent (Invitrogen Ltd # 11668-019). After a further 48hrs (72 hrs after the original transfection), cell viability was assessed using Alamar Blue, and Ai-40 levels were assessed using an A1-40 ELISA (WAKO). A non-targeting siRNA (NTC/scrambled) was used as a negative control. The mock and NTC controls were included in each row of the cell culture plate to account for any row variations. This is why more than one mock control and one NTC control are included in the results.

The rescue validation was run twice. The results of rescue validation experiment 1 are shown in figures 1 Ia and 1 lb and the results of the rescue validation experiment 2 are shown in figures 12a and 12b.

LCAT siRNAs alone caused a decrease in A1-40 relative to cell viability.

Overexpressing LCATmut#1#2-V5 caused an increase in Al-40 levels in the absence of siRNAs, and in the presence of LCAT siRNA #1 and #2 this mutant rescued the decrease in Al-40 production usually observed with these siRNAs.

Thus, this example demonstrates that the decrease in A1-40 levels caused by anti-LCAT siRNAs can be rescued by overexpression of an siRNA resistant LCAT variant.

In the first validation experiment, in the presence of a control siRNA, overexpression of LCATmut#1#2-V5 causes an increase in A1-40. This provides further evidence that LCAT can regulate A31-40 levels. Differences in transfection levels may explain the lack of Aj31-40 increase in the second experiment.

The Western blot demonstrates that LCATmut#1#2 is resistant to LCAT siRNAs #1 and #2 (see figures lib and 12b).

Example 4 Pharmacological validation Two pharmacological inhibitors of LCAT are described in the literature -5,5-Dithiobi s-(2-ni trobenzoic acid) (DTNB) and p-chloromercuriphenylsulfonic acid (PCMPS). DTNB and PCMPS are well-known sulfhydryl inhibitors (See William S. Harris and Alan Rayford. "LCAT inhibitors interfere with the enzymatic determination of cholesterol and triglycerides", Lipids, Vol 25, No. 6, June, 1990).

ELLIN cells were treated overnight with the LCAT inhibitors DTNB and PCMPS.

After this time, cell viability was assessed using Alamar Blue, and A11-40 levels were assessed using an HTRF assay (CisBIO). DTNB and PCMPS reduce A31-4O levels in a dose responsive manner (see figures 13 and 14). The results were expressed as "%dF", which is representative of A1-40 levels. %dF = (100*(ratio sample) -ration blank)/ratio blank i.e. %dF is a value for Af31-40 levels that have had the background subtracted and are normalised to the background.

Example 5 LCAT pathway analysis One of the purposes of analysing the LCAT pathway was to provide further evidence that LCAT is involved in regulating Al1-40 levels because if LCAT regulates A131-40 levels, then other genes that either regulate LCAT itself or modify LCAT substrates, should also regulate A1-4O levels.

A list of 50 genes in a potential "LCAT pathway" was compiled. These genes were not present within the originally screened siRNA libraries described in Example 1.

The genes in the list were chosen either because they have been implicated in the direct regulation of LCAT activity (in at least one primary publication between 2000 and 2008), or because they directly process the LCAT substrates lecithi n/phosphati dylcholine or cholesterol, or other closely related metabolites (pathway information was obtained from the KEOG pathway for glycerophospholipid metabolism http://www.genon-ie.ad.j p/dbget-bin/show....pathway?hsa00564+393 1).

Figures 15a and 15b illustrates the predicted roles of certain genes in the LCAT pathway and hence how they influence AJ31-40 levels. Whether or not these predictions were correct provided a further means of confirming if the LCAT pathway is involved in regulating Al) levels.

Three siRNAs targeting each of the fifty genes potentially identified as being in the LCAT pathway were obtained from Qiagen and resuspended as 2tM stocks. The siRNA sequences for the target genes that were subsequently shown to regulate Al)1- 40 levels are shown in table 1. The siRNAs were screened against A1-40 production in BE2C cells, at a final concentration of 2OnM (5x lower than that used in the original Dharniacon screen). In all cases, ELL1N cells were transfected with Qiagen siRNAs targeting the genes indicated. After 72 hours, the media was harvested. Cell viability was assessed using Alamar blue and A31-4O levels were measured using the HTRF assay (CisBIO).

Genes were classified as hits if two or more siRNAs altered A31-4O production.

From the initial screen, eleven new gene targets were identified from the putative LCAT pathway as potential regulators of A131-40 levels. This is a hit rate of 22% which is significantly higher than would be expected from previous studies carried out with Qiagen siRNAs (approximately 5% obtained in a previous study using Qiagen siRNAs to investigate Af1-40 levels in ELL1N cells). Nine of these eleven target genes altered A131-40 levels in a manner that would have been predicted based on our knowledge of the role of LCAT in regulating A3 levels and an understanding from the literature of the relationship of these nine genes to LCAT (seven of these nine genes are listed in table 2). The results illustrate that the following genes in the LCAT pathway regulate A1 activity: LYPLA2, HNIF1/TCF1, PHOSPHO1, PEMT, HMGCR, SP3 and PCYTIA (see figures 16-19). This pathway analysis data provides strong evidence for a role for the LCAT pathway in regulating A levels.

LYPLA2 Knock out of LYPLA2 resulted in an increased level of A31-40 (see figure 16a).

Anti-LYPLA2 siRNAs do not appear to alter full-length APP levels in ELL1N cells in comparison to mock transfected cells, when taking into account the slight differences in protein loading as assessed by ponceau stain for total protein levels (see figure 16b).

HNF 1 A/TCF1 HNFIA/TCFI knockout mice have increased expression of LCAT. Decreasing TCF1 using siRNAs might therefore be predicted to increase LCAT, and thus A3 levels, which is what was observed (see figure 17a).

siRNAs against HNFI increase full-length APP and APP c-terminal fragment (CTF) levels in ELL1N cells in comparison to mock transfected cells (see figure 17c). This correlates well with the increase in A31-40 observed using these siRNAs.

PHOSPHO 1 siRNAs against PHOSPHO1 increase full-length APP and APP c-terminal fragment (CTF) levels in IELL1N cells in comparison to mock transfected cells (see figure 17c).

This correlates well with the increase in Af31-40 observed using these siRNAs (see figure 17b).

PEMT

See figure 15a for the predicted role of PEMT in the LCAT pathway and hence the predicted effect on A31-4O levels. Knockout of PEMT resulted in a decrease in A13 levels (see figure 18a).

1-IMGCR HIMGCR is a key enzyme involved in cholesterol biosynthesis (cholesterol is an LCAT substrate). See figure 15b for the predicted role of HIvIGCR in the LCAT pathway and hence the predicted effect on A31-40 levels. Knockout of HMGCR resulted in an increase in A levels (see 18b). SP3

SP3 is a transcription factor that binds to the LCAT promoter. SP3 functions as a dose dependent repressor of SP1 mediated LCAT transcriptional activation Hoppe KL and Francone AL. SP3 should therefore decrease LCAT levels. siRNA targeted SP3 might therefore be predicted to increase LCAT, and thus increase Af31-40 levels.

This is the result that is observed. Correlating with this, the two anti-SP3 siRNAs that increase A31-40 levels (SP_1 and SP5) both lead to an increase in full length APP (see figures 18c and 18d).

PCYTIA

See figure 15a for the predicted role of PCYT1A in the LCAT pathway and hence the predicted effect on Aj31-40 levels. Knock out of PCYTIA decreased A13 levels (see figure 19a).

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

s1RNA Name Refseq No. sIRNA Target Sequence HNF1 AITCFI_1 NM_000545 CAGGACAAGCATGGTCCCACA HNFI A/TCFL2 NM_000545 GCGAGTGGCACG'ITI'AlTFAA HNFI AITCFI _4 NM_000545 CAGCCGAGCCATGG1TI'CTAA PHOSPHOI_ I NM_I 78500 TACAACTATAAAGGAGGTGAA PHOSPHOI _2 NM_I 78500 CGCCAACATGTGCAAGCACAA PHOSPHO1 _3 NM_ 178500 CCGTCCCTATCTATTCAGTTA PEMT_I NM_007 169 NM_148 172 NM_148 173 CACATFCCCA11'CACCAATAA PEMT2 NM_007 169 NM_148 172 NM_148 173 CTACATAGTGGCFCTCCFATA PEMT_3 NM_007169 NM_148172 NM_148173 ACCTGGCCTGCTACTCTCTAA HMGCR_I NM_000859 CAAG1TATTACCCTAAG1TFA HMGCR_2 NM_000859 CCGAGCcTAATGAAAGGGAAA HMGCR_3 t4M_000859 GAGCTFG'Il'GTGAGAATGll'A SF3_I NM_001017371 NM_0031 11 cTGCGCGAGATGATAC1T1'GA SF3_5 NM_0OI 017371 NM_003 111 CGGG'm'CTCCTGATATFAAT SF3_S NM_0010 17371 NM_003 III CAGCCTGTTGTACAGCATCTA LYPLA2_8 NM_007260 CAGGGTCCAGTI'CAAGACATA LYPLA2_9 NM_007260 CAAGGCCTTGA1TGAGCATGA LYPLA2_I 0 NM_007260 AAGCTGCTGCCTCCTGTCTAA PCYTI A_5 NM_00501 7 CACGGTGATGAACGAGAATGA PCYTI A_6 NM_005017 CACGCCCGAGCTCTGATGCAA PCYTI A7 NM_0050 17 CACCCGAA11'GTGCGGGATFA

Table 1

ene Name Refseq No. ffect of siRNA;iRNAs altering Effect on APP PEMT M._007 169 Decreases A U3 (#1#3) 4o change in APP levels CYT1A 4M_0050I7)ecreases A3 U3 (#5#6) 4o change in APP levels XPLA2 JM_00726O Increases A 3/3 (#8#9#10) o change in APP levels INFL/TCFI 4M_000545 Increases A 3/3 (#l#2#4) Increase in FL-APP and CTFs HOSPHO1 4M_178500 Increases A 3/3 (#I#2#3) Increase in FL-APP and CTFs IMGCR NM_000859 Increases A13 a/3(#J#3) Increase in FL-APP and CTFs SP3 NM_001017371 Increases A 3/3 (#I#5#8) 1#5 strongly 1 FL and CTFs.

Table 2

Claims

Claims 1. A method of screening a candidate agent for its ability to reduce the activity of lecithin cholesterol acyl transferase (LCAT), phosphatidylethanolamine methyltransferase (PEMT) and/or phosphate cytidylyltransferase 1, choline, alpha (PCYT1A) or increase the activity of phosphatase orphan 1 (PHOSPHOI), 3-hydroxy- 3-methylgi utaryl-coenzyme A reductase (HMGCR), specificity protein 3 transcription factor (SP3), lysophospholipase II (LYPLA2) and/or hepatic nuclear factor lA//transcription factor 1 (1-INFIA/TCFI), the method comprising determining if the activity of LCAT, PEMT and/or PCYTIA is reduced or if the activity of PHOSPHO1, HMGCR, SP3, LYPLA2 or 1-INF1AJTCFI is increased, in the presence of a candidate agent.
2. The method of claim 1, wherein the method comprises contacting a LCAT, PEMT, PCYT1A, PHOSPHO1, HMGCR, SP3, LYPLA1 or HNFIA/TCFI substrate with LCAT, PEMT, PCYT1A, PHOSPHO1, H1v1GCR, SP3, LYPLA! or HNFIA/TCFI in the presence of a candidate agent.
3. The method of claim 1 or claim 2, wherein the method comprises contacting a host cell which expresses LCAT, PEMT, PCYT1A, PHOSPHO1, HMGCR, SP3, LYPLA1 and/or HNFIAITCFI with a candidate agent and determining if the activity of LCAT, PEMT and/or PCYT1A is reduced or if the activity of PHOSPHO1, HMGCR, SP3, LYPLA1 and/or HNF1AJTCF1 is increased..
4. A method, as claimed in claim 3, wherein the host cell is prokaryotic or eukaryotic.
5. A method, as claimed in cell 4, wherein the host cell is a yeast cell.
6. A method, as claimed in any one of claims 3 to 5, wherein the host cell further comprises nucleic acid encoding a reporter gene, which is expressed as a fusion protein with expression of LCAT, PEMT, PCYTIA, PHOSPHOI, HMGCR, SP3, LYPLAI orHNF1AITCF.
7. The method, as claimed in any one of claims 1 to 6, wherein the candidate agent is a synthetic or a natural molecule.
8. A genetic construct comprising a nucleic acid sequence encoding LCAT, PEMT, PCYT1A, PHOSPHO1, HIMGCR, SP3, LYPLA2 or HNF1AITCFI under the control of a constitutive or inducible promoter, suitable for expression of LCAT, PEMT, PCYTIA, PHOSPHO1, HMGCR, SP3, LYPLA2 or HNF1A/TCF1.
9. A host cell which comprises the genetic construct of claim 8.
10. A candidate agent which reduces the activity of LCAT, PEMT andlor PCYTIA or increases the activity of PHOSPHO, IHIMGCR, SP3, LYPLA2 and/or HNFIAITCF1 as identified by the method of any one of claims 1 to 7
11. A method of treating a patient who suffers or is predisposed to suffer from Alzheimer's Disease, the method comprising screening a candidate agent as claimed in any one of claims I to 7, identifying an agent that reduces the activity of LCAT, PEMT and/or PCYT1A or increases the activity of PHOSPHO1, HMGCR, SP3, LYPLA2 and/or HNF1AJTCF1 and administering the agent to the patient.
12. A method of treating a patient who suffers or is predisposed to suffer from Alzheimer's Disease by administration of a therapeutic agent, wherein the therapeutic agent is an agent which reduces the activity of LCAT, PEMT and/or PCYTIA or increases the activity of PHOSPHOI, HMGCR, SP3, LYPLA2 and/or HNF1AITCF1.
13. An agent which reduces the activity of LCAT, PEMT and/or PCYT1A or increases the activity of PHOSPHOI, HMGCR, SP3, LYPLA2 and/or H1'JF1AITCF1 for use in treating Alzheimer's Disease.
14. Use of an agent which reduces the activity of LCAT, PEMT and/or PCYTIA or increases the activity of PHOSPHO1, HIMGCR, SP3, LYPLA2 and/or HNF1AITCFI in the manufacture of a medicament for treating Alzheimer's Disease.
15. A pharmaceutical composition comprising an agent which reduces the activity of LCAT, PEMT and/or PCYT1A or increases the activity of PHOSPHO1, HMGCR, SP3, LYPLA2 and/or HNFIAITCFI.