The Treatment of Protein Aggregation Diseases

Abstract

Compositions, methods and systems for the treatment of a protein aggregation disease including, but not limited to Alzheimer's disease (AD), Parkinson's disease (PD), Dementia with Lewy Bodies (DLB), Huntington's disease (HD), Amylotrophic lateral sclerosis (ALS, which results from degeneration of the upper and lower motor neurones and affects the voluntary muscle system), Progressive Supranuclear Palsy (PSP), Type 2 Diabetes and Multiple systems atrophy (MSA).

Claims

1. A red blood cell preparation derived from one or more of the following: i) a donor or donors; ii) the subject's red blood cells or the donor or donor's red blood cells that have been treated ex vivo to reduce the content of protein oligomers and/or aggregates; iii) stem cells or other mononuclear cells; and iv) from a xenotransfusion source, wherein the level of oligomers or aggregates of proteins in the red blood cell preparation has been measured and shown to be at a reduced level.

2. (canceled)

3. A red blood cell preparation as claimed in claim 1 wherein the level of protein oligomers or aggregates in the administered red blood cells has been measured with an analytical method and shown to be at a reduced level below the detection limit of the analytical method.

4. A method of treating a protein aggregation disease in a subject comprising administering red blood cells derived from one or more of the following: i) a donor or donors ii) the subject's red blood cells or the donor or donor's red blood cells that have been treated ex vivo to reduce the content of protein oligomers and/or aggregates iii) derived from stem cells or other mononuclear cells iv) from a xenotransfusion source wherein the level of protein oligomers or aggregates in the administered red blood cells has been measured and shown to be at a reduced level.

5. The method according to claim 4, wherein the red blood cells are derived from one or more of i) a donor or donors under the age of 50 years; ii) the subject's red blood cells that have been treated ex vivo to remove toxic oligomers and/or aggregates; iii) derived from stem cells or other mononuclear cells; and iv) from a xenotransfusion, wherein an assay is used to confirm that the level of toxic oligomers or aggregates in the red blood cells to be administered is at or below the detection limit of the analytical method and the analytical method is used subsequently to detect an increase in the levels in the blood of the subject above a pre-defined threshold thereby triggering a repeat of the therapeutic apheresis process.

6. A method as claimed in claim 4 wherein the level of protein oligomers or aggregates in the administered red blood cells has been measured with an analytical method and shown to be at a reduced level below the detection limit of the analytical method.

7. A red blood cell preparation according to claim 1, wherein the oligomeric or aggregated proteins comprise any one or more of the following: Abeta, alpha synuclein, DJ-1 (also known as Park7), tau, superoxide dismutase (SOD), or IAPP.

8. A method of treating a protein aggregation disease comprising removing aggregated proteins from the surface of red blood cells.

9. The method as claimed in claim 8, wherein the removal of the aggregated proteins from the surface of the red blood cells takes place ex vivo.

10. A method for removing aggregated proteins from the surface of a red blood cell comprising contacting a subject's red blood cells with means to remove protein aggregates from the surface of a cell.

11. The method as claimed in claim 10, wherein the means to remove the protein aggregates from the surface of a red blood cell comprises an antibody, an antibody fragment and/or synthetic antibody capable of binding to the aggregated protein of interest.

12. The method as claimed in claim 11, wherein the antibody, antibody fragment and/or synthetic antibody is immobilised immobilized on a substrate.

13. The method as claimed in claim 12, wherein the substrate comprises any one or more of the following: membrane filters, magnetic beads, non-magnetic beads or monolithic high surface area substrates.

14. The method as claimed in claim 13, wherein the substrate comprises magnetic and/or non-magnetic beads having a diameter in the range 0.1 μm to 150 μm.

15. The method as claimed in claim 14, further comprising separating the red blood cell from the aggregated protein.

16. The method as claimed in claim 15, comprising the subsequent step of reintroducing the red blood cells into a subject.

17-20. (canceled)

Description

[0052] The present invention will now be described, by way of example only, with reference to the following Examples and Figures.

[0053] FIG. 1 shows oligomer depletion with A11 antibody prior to using the full assay protocol.

[0054] FIG. 2 shows the levels of oligomeric Abeta in whole blood samples from healthy normal controls and from Alzheimer's Disease (AD), Multiple Systems Atrophy (MSA) and Parkinson's Disease (PD) patients. The X axis shows age and gender of the controls and patients, CS' indicates a sample from an age matched spouse. Y axis shows the signal obtained from the oligomeric Abeta assay protocol using a Europium label as relative fluorescence units (RFU) as measured by an LFB-Wallac time-resolved fluorimeter; for the oligomeric ASN study the Y axis shows the signal obtained using an HRP enzyme conjugate, in this case the signal is reported as optical density as measured in a Biotek Inc. colorimetric microplate reader.

[0055] FIG. 3a shows an analysis of the control samples as a box and whisker plot and confirms that there is a rise in the median level of oligomeric Abeta with advancing age.

[0056] FIG. 3b shows a box and whisker plot with the added data from the AD patients with advancing age and also includes the MSA and PD patients.

[0057] FIG. 3c shows a box and whisker plot with the control samples as well as the AD, MSA and PD patients.

[0058] FIG. 4a shows the results from a Ficol gradient separation of whole blood from two AD patients and shows the levels of oligomeric Abeta in the three fractions; plasma, buffy coat (leukocytes) and red cells.

[0059] FIG. 4b shows the results from a Ficol gradient separation of whole blood from two MSA patients and shows the levels of oligomeric Abeta in the three layers; plasma, buffy coat (lymphocytes) and predominantly red cells.

[0060] FIG. 5 shows the levels of oligomeric ASN in whole blood samples from healthy normal controls and from AD, MSA and PD patients. The X axis shows age and gender of the controls and patients.

[0061] FIG. 6 shows an analysis of the control samples and AD and MSA as a box and whisker plot and confirms that there is no observable rise in the median level of oligomeric ASN with advancing age whilst AD, MSA and PD patients exhibited increases in oligomeric ASN.

[0062] FIG. 7a shows the results from a Ficol gradient separation of whole blood from two AD patients and shows the levels of oligomeric ASN in the three layers; plasma, buffy coat (lymphocytes) and predominantly red cells.

[0063] FIG. 7b shows the results from a Ficol gradient separation on whole blood from two MSA patients and shows the levels of oligomeric ASN in the three layers; plasma, buffy coat (lymphocytes) and predominantly red cells.

EXAMPLES

Example 1. Oligomer Depletion with all Antibody

[0064] Abeta 1-42 and ASN were aggregated according to the methods described in WO2017067672. Protein A-coated magnetic beads (GE Healthcare Inc.), 37-100 μM in diameter, were derivatised with A11 antibody from Thermo Fisher Scientific Inc (2 μl of A11 antibody added to 50 μl of bead suspension) and then added to 1 ml of 100 μg/ml aggregated protein solution in PBS buffer pH 7.5. The mixture was then placed on a rotating incubator for 1 h at room temperature. The tubes were transferred to a magnetic separator and the depleted supernatant was collected for further processing in the full assay protocol described in WO2017067672. A11 has specificity for the oligomeric forms of both Abeta and ASN but does not distinguish between these proteins, most likely the antibody recognizes the conformation of the peptide backbone in an oligomer. FIG. 1 shows that the signal in the assay was reduced to background after treatment with A11-coated beads thus indicating that the full assay protocol was measuring the oligomeric forms of these proteins only.

Example 2

[0065] The full assay protocol described in WO2017067672 was applied to frozen whole blood samples collected from healthy normal controls and from Alzheimer's Disease (AD), Multiple Systems Atrophy (MSA) and Parkinson's Disease (PD) patients. Controls were obtained from healthy volunteers and were collected with full ethical permission by the Liverpool BioInnovation Biobank, UK. Control blood samples were also supplied by Tissue Solutions, Glasgow, UK. Further control samples from age matched spouses were supplied by Salford Royal Infirmary, UK. Results from the Abeta assay protocol are shown in FIG. 2 and the ASN assay protocol are shown in FIG. 5.

Example 3

[0066] A Ficoll (GE Healthcare) gradient separation of fresh whole blood (not frozen) from AD and MSA patients was carried out using the manufacturer's instructions. An aliquot of 1 ml volume was taken from each layer within the gradient and analysed in the full oligomeric protein assay protocol. FIGS. 4a and 4b show the results from the full assay protocol on each layer from the gradient.