PRODUCTS AND METHODS ASSOCIATED WITH MULTIPLE SCLEROSIS AS A TRANSMISSIBLE PROTEIN MISFOLDING DISORDER
20200229407 ยท 2020-07-23
Inventors
Cpc classification
A61K31/436
HUMAN NECESSITIES
A01K67/0275
HUMAN NECESSITIES
A61K31/473
HUMAN NECESSITIES
A61K31/737
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
A61K31/454
HUMAN NECESSITIES
A01K2207/10
HUMAN NECESSITIES
International classification
A61K31/454
HUMAN NECESSITIES
A61K31/473
HUMAN NECESSITIES
A61K31/737
HUMAN NECESSITIES
Abstract
Described are methods and products related to the identification of multiple sclerosis (MS) as a transmissible protein misfolding disorder. Data is presented to support the position that the transmissible protein is an abnormal prion protein conformer (PrP.sup.MS). Methods are described for identifying a subject having, or at risk of developing, multiple sclerosis (MS) based on determining the presence or absence of PrP.sup.MS in a sample from the subject. The presence of the abnormal prion protein conformer in the sample is indicative of the subject having MS or an increased risk of developing MS. Also described are therapeutics methods for the treatment of MS as well as cell cultures, non-human animal models and biological samples thereof useful for the study of MS.
Claims
1. A method of identifying a subject having, or at risk of developing, multiple sclerosis (MS), the method comprising: detecting the presence or absence of an abnormal prion protein conformer (PrP.sup.MS) in a sample from the subject, wherein the presence of the abnormal prion protein conformer in the sample is indicative of the subject having MS or an increased risk of developing MS, wherein PrP.sup.MS is a transmissible, proteinase K-sensitive conformer of PrP.
2. The method of claim 1, wherein the abnormal prion protein conformer is absent in a control sample from a subject without MS.
3. The method of claim 1, wherein the abnormal prion protein conformer forms deposits of -sheet rich amyloid.
4. The method of claim 1, wherein the abnormal prion protein conformer causes transmission of MS pathology to a test subject when injected intracerebrally into the test subject.
5. The method of claim 4, wherein the abnormal prion protein conformer causes the test subject to develop brain atrophy, amyloid plaques, demyelination, abnormal behavior and/or ventriculomegaly.
6. The method of claim 4, wherein the test subject is a transgenic mouse expressing or overexpressing prion protein (PrP), optionally human prion protein.
7. The method of claim 1, wherein pronase E digestion of PrP.sup.MS generates smaller molecular weight fragments of prion protein that are resistant to further digestion and wherein PrP.sup.MS is more resistant to pronase E digestion relative to prion protein from a control sample from a subject without MS.
8. The method of claim 1, wherein the sample is a tissue sample, a cerebrospinal fluid (CSF) sample, or a blood sample.
9.-10. (canceled)
11. The method of claim 8, wherein the tissue sample is a brain tissue sample and the method comprises detecting the abnormal prion protein conformer in the brain tissue sample in extracellular space, around vessels, oligodendrocytes, oligodendrocyte precursor cells, microglia, astrocytes, neurons, cortex and/or white matter.
12.-13. (canceled)
14. The method of claim 1, wherein detecting the presence or absence of the abnormal prion protein conformer in the sample comprises spectral analysis.
15.-17. (canceled)
18. The method of claim 1, wherein detecting the presence or absence of the abnormal prion protein conformer in the sample comprises RT-QuIC, immunohistochemistry or Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) and/or increasing the number of abnormal prion protein conformers in the sample using protein-misfolding cyclic amplification (PMCA).
19.-39. (canceled)
40. A non-human animal model of multiple sclerosis (MS), produced by administering abnormal prion protein conformer (PrP.sup.MS) to a non-human animal, wherein PrP.sup.MS is a transmissible, proteinase K-sensitive conformer of PrP.
41. (canceled)
42. The non-human animal model of claim 40, wherein the non-human animal is a non-human primate or a rodent.
43. The non-human animal model of claim 40, wherein the non-human animal develops one or more signs or symptoms of MS after administration of the abnormal prion protein conformer, wherein the signs or symptoms of multiple sclerosis are selected from the presence of amyloid plaques, brain atrophy, demyelination, axonal damage, ventriculomegaly and motor/pain perception/cognitive/visual abnormalities.
44. (canceled)
45. The non-human animal model of claim 40, produced by administering abnormal prion protein conformer (PrP.sup.MS) to the non-human organism by intracerebral or intraperitoneal injection.
46. The non-human animal model of claim 40, produced by administering to the non-human organism a brain homogenate, spinal chord homogenate, CSF or other biological sample comprising abnormal prior protein conformer (PrP.sup.MS), optionally wherein the brain homogenate, spinal chord homogenate, CSF or other biological sample is from a subject with MS.
47. The non-human animal model of claim 40, wherein the non-human animal is a transgenic animal that overexpresses prion protein.
48.-52. (canceled)
53. A method of identifying candidate compounds for the detection or treatment of multiple sclerosis (MS), the method comprising: (a) administering a test compound to the non-human animal model of claim 40; and (b) detecting a biological effect of the test compound on the non-human animal model.
54. (canceled)
55. The method of claim 53, wherein the biological effect is a reduction in the presence of abnormal prion protein conformer and/or amyloid-like deposits in the non-human model organism relative to a control, or an increase or decrease in one or more signs or symptoms of MS, wherein the signs or symptoms of MS are selected from the presence of amyloid plaques, demyelination, brain atrophy, ventriculomegaly and abnormal animal behavior.
56.-73. (canceled)
74. A method of producing a non-human animal model of multiple sclerosis (MS), the method comprising administering abnormal prion protein conformer (PrP.sup.MS) to the non-human animal, wherein PrP.sup.MS is a transmissible, proteinase K-sensitive conformer of PrP, wherein the non-human animal develops one or more signs or symptoms of MS after administration of PrP.sup.MS.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Embodiments of the invention will now be described in relation to the drawings in which:
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DETAILED DESCRIPTION OF THE INVENTION
[0040] The inventors have surprisingly determined that subjects with multiple sclerosis (MS) exhibit abnormal deposits of prion protein (PrP) and that MS pathology is associated with a progressive transmissible prion protein folding disorder.
[0041] Without being limited by theory, it is believed that MS is caused by misfolding of normal prion protein (PrP.sup.C) into an aggregation-prone conformer, PrP.sup.MS, which accumulates in the brain of MS patients and causes dysregulation of myelinic GluRs and non-immune demyelination. The accumulation of pathological prion may induce chronic dysregulation of the axo-myelinic synapse culminating in progressive disease. Previously it was not known that abnormal PrP could be detected in subjects with MS nor has there been any biological data demonstrating disease transmission consistent with MS being a prionopathy. In contrast, a commonly accepted view is that MS is a primary autoimmune disease associated with genetic and/or environmental risk factors.
[0042] As set out in Example 1 and
[0043] As set out in Example 2 and
[0044] Furthermore, as shown in
[0045] As set out in Example 6 and
[0046] Additional experiments shown in
[0047] Together, the data presented herein supports the position that MS is a weakly transmissible protease-sensitive prionopathy and that the etiological agent of MS is a misfolded prion conformer identified herein as PrP.sup.MS.
[0048] In one embodiment, there is provided a method of identifying a subject having, or at risk of developing, multiple sclerosis (MS). In one embodiment, the method comprises determining the presence or absence of an abnormal prion protein conformer (PrP.sup.MS) in a sample from the subject. In one embodiment, the presence of the abnormal prion protein conformer in the sample is indicative of the subject having MS or an increased risk of developing MS. In one embodiment, the PrP.sup.MS conformer is absent in a control sample from a subject without MS. Optionally the control sample is matched for age, gender, ethnicity or genetic background with the subject who provides the sample.
[0049] As used herein, multiple sclerosis (MS) refers to a neurological disability characterized by formation of lesions or plaques in the central nervous system, inflammation, and/or the destruction of myelin sheaths of neurons leading to autonomic, visual, motor, and/or sensory problems. MS may be diagnosed on the basis of clinical presentation, medical imaging and/or laboratory testing. In one embodiment, MS may be diagnosed based on the McDonald criteria, including the dissemination of lesions in space and time. Optionally, a diagnosis of MS may include the use of magnetic resonance imaging (MRI) to show the existence of demyelinating lesions, that they occur in different areas of the nervous system and/or that they accumulate over time.
[0050] The term subject as used herein refers to any member of the animal kingdom. In one embodiment the subject is a mammal, such as a human. Optionally, the subject is a human presenting with one or more signs or symptoms of MS. In one embodiment, the subject is non-human animal, optionally a non-human primate. In one embodiment, the subject is a rodent, such as a mouse, hamster or a bank vole.
[0051] In one embodiment, determining the presence or absence of the abnormal prion protein conformer comprises detecting the presence or absence of the abnormal prion protein conformer in the sample.
[0052] Various methods may be used to detect the PrP.sup.MS conformer described herein. For example, the PrP.sup.MS conformer can be distinguished from cellular prion protein and/or prion proteins that are not associated with MS pathogenesis. In one embodiment, the PrP.sup.MS conformer is detected in vitro or detected in vivo using a test subject. In one embodiment the PrP.sup.MS conformer forms deposits of -sheet rich amyloid.
[0053] The PrP.sup.MS conformer may also be detected using biochemical techniques that distinguish between various forms of the PrP protein. For example, in one embodiment the PrP.sup.MS conformer is detected by isolating or purifying prion proteins in a sample, subjecting the sample to digestion with a protease and detecting the protein fragments generated by protease digestion. In one embodiment, pronase-E digestion of the PrP.sup.MS conformer generates smaller molecular weight fragments of prion protein that are resistant to further digestion, relative to pronase-E digestion of prion protein from a control sample from a subject without MS which almost completely digests the prion protein. In one embodiment, proteinase K or thermolysin digestion of the PrP.sup.MS conformer generates smaller molecular weight fragments of prion protein that are resistant to further digestion, relative to proteinase k and/or thermolysin digestion of prion protein from a control sample from a subject without MS which almost completely digests the prion protein. In one embodiment, PrP.sup.MS complexes are stabilized using Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) prior to detection, such as by immunoassay, precipitation with PTA, and/or by spectral analysis. In one embodiment, the immunoassay comprises a Western blot to detect large molecular weight aggregates that differ in size from controls.
[0054] In one embodiment, the PrP.sup.MS conformer may be detected in vivo using a test subject. As shown in the Examples, the PrP.sup.MS conformer causes transmission of MS-like pathologies to a test subject when administered to the test subject. In one embodiment, the PrP.sup.MS conformer causes transmission of MS-like pathologies to a test subject when injected intracerebrally into the test subject. In one embodiment, the test subject is a transgenic animal expressing human prion protein. In one embodiment, the test subject is a bank vole or mouse. In one embodiment, the test subject is a mouse, optionally a tg650 mouse or tga20 mouse. In one embodiment the presence or absence of PrP.sup.MSconformer in the sample administered to the test subject is determined by detecting the presence or absence of lesions or plaques containing abnormal protein aggregates in the test subject. In one embodiment, the presence or absence of PrP.sup.MS conformer in the sample administered to the test subject is determined by detecting MS-like pathologies in the test subject such as brain atrophy, amyloid plaque, demyelination and/or ventriculomegaly. In one embodiment, the presence of formic acid-resistant PrP aggregates in the test subject is indicative of the PrP.sup.MS conformer in the sample.
[0055] As shown in the Examples, abnormal prion protein conformers associated with MS have been detected in brain tissue and cerebrospinal fluid. Furthermore, as shown in
[0056] In one embodiment, the tissue sample is a brain tissue sample and the method comprises detecting the abnormal prion protein conformer in the brain tissue sample in extracellular space, around vessels, oligodendrocytes, myelin, neurons, cortex and/or white matter.
[0057] In one aspect, the presence or absence of the PrP.sup.MS conformer in the sample is detected using spectral analysis. For example, the PrP.sup.MS conformer may be detected using fluorescent spectroscopy and/or absorption spectroscopy and probes known to bind prion protein aggregates. In one embodiment the spectral analysis comprises:
[0058] (a) contacting the sample with a conformationally sensitive probe that binds to the abnormal prion protein conformer;
[0059] (b) generating a fluorescence emission spectrum or absorption spectrum of the conformationally sensitive probe bound to the abnormal prion protein conformer in the sample,
[0060] (c) comparing the fluorescence emission spectrum or absorption spectrum to one or more reference spectra.
[0061] In one embodiment, the reference spectra are representative of spectra from a subject or group of subjects known to have MS and correspondence between the reference spectra and the fluorescence emission spectrum or absorption spectrum is indicative that the subject has MS or an increased likelihood of developing MS. In another embodiment, the reference spectra are representative of spectra from a subject or group of subjects known not to have MS and correspondence between the reference spectra and the fluorescence emission spectrum or absorption spectrum is indicative that the subject does not have MS or an has a decreased likelihood of developing MS.
[0062] Conformationally sensitive probes useful for the spectral analysis of PrP conformers are known in the art. For example, in one embodiment the conformationally sensitive probe is selected from K114, Congo Red, a Congo Red derivative, X34, BSB, FSB, IMSB, Chrysamine-G, methoxy-X34, methoxy-X04, thioflavin-T, thioflavin-S, Pittsburgh compound B, thiazine red R, auramine-O, p-FTAA or a luminescent conjugated polythiophene (LCP) or luminescent conjugated oligothiophene (LCO) related to p-FTAA.
[0063] In another embodiment, the presence or absence of the PrP.sup.MS conformer in the sample may be detected using binding agents such as antibodies and/or immunohistochemical methods.
[0064] In one embodiment, the method comprises fixation of the sample, optionally a blood sample or a tissue sample, using a fixative. In one embodiment, the fixative is formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, methanol, acetone, or Hepes-glutamic acid-buffer mediated organic solvent protection effect (HOPE). In one embodiment, the fixing agent is formalin. In one embodiment, the fixed sample is heated, optionally to between about 80 C. and 98 C. for between about 10 and 30 minutes. In one embodiment, the sample is heated to 90-95 C. in citrate buffer for between about 10 and 20 minutes. In one embodiment, the fixed sample is treated with formic acid to selectively degrade PrP.sup.C. In one embodiment, the fixed sample is treated with 70-100%, optionally 95-98% formic acid for between about 5 minutes and 30 minutes, optionally between 8 minutes and 12 minutes or about 10 minutes, followed by extensive washing in buffer. In one embodiment, the method comprises contacting the sample with an antibody that selectively binds to prion protein and detecting the antibody, optionally the 3F4 antibody.
[0065] In one embodiment, the sample from the subject may be treated to increase the amount of PrP.sup.MS conformer in the sample prior to detection. For example, in one embodiment, the methods described herein include the use of protein-misfolding cyclic amplification (PMCA) prior to detecting the presence or absence of the PrP.sup.MS conformer in the sample. In one embodiment, methods described herein include the use of photo-induced crosslinking methods (e.g. PICUP) prior to detecting the presence or absence of the PrP.sup.MS conformer in the sample.
[0066] In one aspect, there is also provided a cell culture produced by contacting one or more cells with a composition comprising brain or spinal cord homogenate from a subject with MS and culturing the one or more cells. In one embodiment, there is provided a cell culture produced by contacting one or more cells with a composition comprising abnormal prion protein conformer (PrP.sup.MS) and culturing the one or more cells. In one embodiment, the resulting PrP.sup.MS cell culture contains one or more copies of the PrP.sup.MS conformer resulting in the pathological aggregation of endogenous prion protein in the cell culture. In one embodiment, cells are brain cells and/or immune cells. In one embodiment, the cells are transgenic and/or recombinant cells that express or overexpression prion protein. In one embodiment, the cells express or overexpress human or bank vole prion protein.
[0067] As used herein the term cell culture refers to the maintenance and/or growth of one or more cells in a culture medium under controlled conditions. In one embodiment, the cells may be cultured as a layer on a substrate. In another embodiment, the cells may be cultured floating in a suspension. In one embodiment the cell culture comprises a plurality of cells of the same type or a plurality of cells of different types. As used herein the term cell culture includes tissue culture. For example, in one embodiment the cell culture is a culture of a plurality of cells from a tissue sample. In one embodiment the cell culture is a cerebellar tissue culture.
[0068] In one embodiment the cells are brain cells such as neurons, astrocytes, oligodendrocytes, oligodendrocyte precursor cells and/or microglia. In one embodiment, the cells are immune cells such as B cells and/or Follicular Dendritic cells, optionally alone or in combination with brain cells. In one embodiment, the cell culture comprises tissue culture. In one embodiment, the cell culture comprises central nervous system (CNS) tissue, optionally a slice culture of CNS tissue. In one embodiment, the cell culture comprises brain tissue. In one embodiment, the cell culture comprises cortical tissue, corpus callosum tissue, cerebellar tissue, ex vivo optic nerve tissue and/or spinal dorsal column/dorsal root ganglion (DRG) tissue. In one embodiment, the cells are transgenic cells. In one embodiment, the cells express or overexpress prion protein, optionally human prion protein. In one embodiment, the cells express or overexpress prion protein that form aggregates with human prion protein, such as prion protein from the bank vole. In one embodiment, the cells are from a stable cell line. In one embodiment, the cells are human glial (oligodendrocytic) cells that overexpress PrP. For example, in one embodiment the cells are from a huPrP-overexpressing MO3.13 cell line.
[0069] In one embodiment, a PrP.sup.MS cell culture as described herein may be obtained by contacting one or more cells with a biological sample from a subject with multiple sclerosis. In one embodiment, the biological sample is a brain homogenate. Optionally, the biological sample is treated to increase the relative concentration of abnormally folded prion protein, such as by the use of phosphotungstic acid (PTA) to precipitate PrP.sup.MS prior to contacting the one or more cells. In one embodiment, the biological sample is treated to stabilize the PrP.sup.MS prion conformer prior to contacting the one or more cells, such as by Photo-Induced Cross-Linking of Unmodified Proteins (PICUP). In one embodiment, the cells used to generate the PrP.sup.MS cell culture are recombinant cells. For example, in one embodiment the cells overexpress prion protein, optionally human prion protein or bank vole prion protein. In one embodiment, the cells used to generate the PrP.sup.MS cell culture are cells from tg650 mice.
[0070] In one embodiment, the PrP.sup.MS cell culture is obtained by contacting one or more cells with a PrP.sup.MS cell culture as described herein or a preparation thereof. For example, in one embodiment the PrP.sup.MS cell culture is obtained by contacting one or more cells with a composition comprising a PrP.sup.MS cell culture that has been lysed and optionally contacted with phosphotungstic acid (PTA) to precipitate PrP.sup.MS prior to contacting the one or more cells.
[0071] In one embodiment, the PrP.sup.MS cell cultures described herein may be used in screening methods for identifying compounds suitable as diagnostic agents for the detection of MS and/or therapeutic agents for the treatment of MS.
[0072] In one embodiment, there is provided a method comprising:
[0073] (a) contacting a PrP.sup.MS cell culture with a test compound; and
[0074] (b) detecting a biological effect of the test compound on the PrP.sup.MS cell culture.
[0075] In one embodiment, the biological effect is a reduction in the presence of abnormal prion protein conformer and/or amyloid-like deposits in the PrP.sup.MS cell culture relative to a control. Various methods, including but not limited to those described herein, may be used for detecting a biological effect resulting from the test compound on the PrP.sup.MS cell culture. For example, in one embodiment detecting the biological effect of the test compound comprises histological, immunohistochemical and/or spectroscopic detection of abnormal prion protein conformer (PrP.sup.MS) and/or amyloid plaques in the PrP.sup.MS cell culture.
[0076] In one embodiment, a test compound that results in a decrease in the presence of PrP.sup.MS conformer and/or amyloid plaques in the PrP.sup.MS cell culture relative to a control is identified as a candidate therapeutic agent for the treatment of MS. In one embodiment, a test compound that results in a modification of cytotoxicity, cell maturity, cell viability and/or cytodegeneration in the PrP.sup.MS cell culture relative to a control is identified as a candidate therapeutic agent for the treatment of MS. In one embodiment, a test compound that results in decreased cytotoxicity, increased cell maturity, increased cell viability and decreased cytodegeneration is identified as a candidate therapeutic agent for the treatment of MS.
[0077] In another aspect, there is provided a non-human animal model of multiple sclerosis (MS). In one embodiment, the non-human animal model of MS is produced by administering brain homogenate or spinal cord homogenate from a subject with MS to the non-human animal, optionally after passaging in one or more non-human animals. In one embodiment, the non-human animal model of MS is produced by administering abnormal prion protein conformer (PrP.sup.MS) to a non-human animal. In one embodiment, the non-human animal develops one or more MS-like signs or symptoms after administration of the abnormal prion protein conformer. For example, in some embodiment, the non-human animal model of MS exhibits one or more MS-like signs or symptoms selected from the presence of amyloid plaques, demyelination, brain atrophy ventriculomegaly, and abnormal animal behavior.
[0078] The non-human animal may be a mammal, optionally a rodent such as a mouse, hamster or bank vole, or a non-human primate. Optionally, the non-human animal may be a transgenic animal that expresses or overexpresses prion protein. In one embodiment, the non-human may be a transgenic animal that expresses or overexpresses human prion protein. In one embodiment, the non-human animal is a tg650 mouse
[0079] Different methods may be used to administer an effective dose of PrP.sup.MS conformer to a non-human animal to produce an animal model of MS. In one embodiment, a composition comprising PrP.sup.MS conformer is administered to the non-human animal by intracerebral or intraperitoneal injection. The composition comprising PrP.sup.MS conformer may be brain homogenate from a subject with MS. Alternatively, the composition may be a PrP.sup.MS cell culture or preparation thereof such as a PrP.sup.MS cell culture treated to increase the concentration of PrP.sup.MS conformers.
[0080] In one embodiment, the non-human animal models of MS described herein may be used in screening methods for identifying compounds suitable as diagnostic agents for the detection of MS and/or therapeutic agents for the treatment of MS.
[0081] In one embodiment there is provided a method comprising:
[0082] (a) administering a test compound to a non-human animal model of MS as described herein; and
[0083] (b) detecting a biological effect of the test compound on the non-human animal model.
[0084] The biological effect may be a reduction in the presence of abnormal prion protein conformer and/or amyloid-like deposits in the non-human model organism relative to a control. Various methods, including but not limited to those described herein, may be used for detecting a biological effect resulting from the test compound on the non-human model of MS.
[0085] For example, in one embodiment detecting the biological effect of the test compound comprises histological, immunohistochemical and/or spectroscopic detection of abnormal prion protein conformer (PrP.sup.MS) and/or amyloid plaques in a sample from the non-human model of MS.
[0086] In one embodiment, the biological effect is an increase or decrease in one or more signs or symptoms of MS in the model animal. For example, in one embodiment, the signs or symptoms of MS are selected from the presence of amyloid plaques, brain atrophy, demyelination, ventriculomegaly, abnormal animal behavior and/or motor/pain perception/cognitive/visual abnormalities. In one embodiment, the biological effect is a reduction in the presence of PrP.sup.MS conformer and/or amyloid-like deposits in the non-human model animal relative to a control animal. In one embodiment, the biological effect is demyelination and/or degeneration, optionally measured through Magnetic Resonance Imaging (MRI), Computerized Tomography (CT) and/or Micro/Nano Computed Tomography (micro-CT and/or nano-CT).
[0087] In one embodiment, a test compound that results in a decrease in the presence of PrP.sup.MS conformer and/or amyloid plaques or a decrease in one or more signs or symptoms of MS in the non-human animal model of MS relative to a control is identified as a candidate therapeutic agent for the treatment of MS.
[0088] In one aspect, the inventors have surprisingly determined that subjects with multiple sclerosis (MS) exhibit abnormal deposits of prion protein (PrP) and that MS pathology is associated with a progressive transmissible prion protein folding disorder. Agents that reduce a level and/or inhibit the formation of the abnormally folded prion protein may therefore be useful for the prevention or treatment of MS.
[0089] Without being limited by theory, it is believed that MS is caused by misfolding of normal prion protein (PrP.sup.C) into an aggregation-prone conformer, PrP.sup.MS, which accumulates in the brain of MS patients and causes dysregulation of myelinic GluRs and non-immune demyelination. The accumulation of pathological prion may induce chronic dysregulation of the axo-myelinic synapse culminating in progressive disease. Previously it was not known that abnormal PrP could be detected in subjects with MS nor has there been any biological data demonstrating disease transmission consistent with MS being a prionopathy. In contrast, a commonly accepted view is that MS is a primary autoimmune disease associated with genetic and/or environmental risk factors.
[0090] Together, the data presented herein supports the position that MS is a weakly transmissible protease-sensitive prionopathy and that the etiological agent of MS is a misfolded prion conformer identified herein as PrP.sup.MS. Agents that reduce the levels of abnormally folded prion protein and/or inhibit the formation of abnormally folded prion protein may therefore be useful for the prevention or treatment of MS. In one embodiment, the agents that reduce and/or inhibit the formation of PrP.sup.MS are listed in Table 1.
TABLE-US-00001 TABLE 1 Agents for the prevention or treatment of MS caused by PrP.sup.MS Agent Quinacrine Pentosan polysulphate (PPS) Tetracycline antibiotics, including doxycycline and minocycline Astemizole Tacrolimus (FK-506) Fluorescent Amyloid dyes (e.g. K114, Congo Red, a Congo Red derivative, X34, BSB, FSB, IMSB, Chrysamine-G, methoxy-X34, methoxy-X04, thioflavin-T, thioflavin-S, Pittsburgh compound B, thiazine red R, auramine-O, pFTAA, or a luminescent conjugated polythiophene (LCP) or luminescent conjugated oligothiophene (LCO) related to p-FTAA. Anle138b Antibodies that bind to PrP.sup.MS Molecular modifications of the agents listed herein with the purpose of improving bioavailability, safety or crossing of the blood brain barrier
[0091] In one embodiment, the agent is quinacrine and is for the treatment and/or prevention of MS. Korth et al. [121] (Proc Natl Acad Sci USA. 2001 Aug. 14; 98(17):9836-41, hereby incorporated by reference) reported that quinacrine inhibited the formation of pathogenic prions (PrP.sup.SC) in cultured cells. In one embodiment, quinacrine may therefore be useful for reducing and/or preventing the formation of PrP.sup.MS conformers.
[0092] In one embodiment, the agent is pentosan polysulphate (PPS) and is for the prevention of MS. Doh-ura et al. [122] (J Virol. 2004 May; 78(10):4999-5006, hereby incorporated by reference) showed that intraventricular application of PPS prolonged the incubation period in transgenic mice infected with scrapie agent. In one embodiment, PPS may therefore be useful for reducing and/or preventing the formation of PrP.sup.MS conformers.
[0093] In one embodiment, the agent is a tetracycline antibiotic, such as doxycycline or minocycline, and is for the prevention of MS. De Luigi et al. [123] (PLoS One. 2008 Mar. 26; 3(3):e1888, hereby incorporated by reference) described the efficacy of tetracyclines in peripheral and intracerebral prion infection. In one embodiment, tetracyclines may therefore be useful for reducing and/or preventing the formation of PrP.sup.MS conformers.
[0094] In one embodiment, the agent is astemizole and is for the treatment and/or prevention of MS. Karapetyan et al. [124] Proc Natl Acad Sci USA. 2013 Apr. 23; 110(17):7044-9, hereby incorporated by reference) identified astemizole as reducing cell-surface PrP and inhibiting prion replication in neuroblastoma cells. In one embodiment, astemizole may therefore be useful for reducing and/or preventing the formation of PrP.sup.MS conformers.
[0095] In one embodiment, the agent is tacrolimus (FK-506) and is for the prevention of MS. Karapetyan et al. [124] Proc Natl Acad Sci USA. 2013 Apr. 23; 110(17):7044-9, hereby incorporated by reference) identified tacrolimus as reducing cell-surface PrP and inhibiting prion replication in neuroblastoma cells. In one embodiment, tacrolimus may therefore be useful for reducing and/or preventing the formation of PrP.sup.MS conformers.
[0096] In one embodiment, the agent is a fluorescent amyloid dye and is for the treatment and/or prevention of MS. Herrmann et al. [125] (Sci Transl Med. 2015 Aug. 5; 7(299):299ra123, hereby incorporated by reference) used structure-based drug design to identify polythiophenes as antiprion compounds. Fluorescent amyloid dyes are known to bind to pathogenic PrP conformers as demonstrated herein and are therefore expected to be useful for reducing and/or preventing the formation of PrP.sup.MS conformers
[0097] In one embodiment, the agent is Anle138b and is for the treatment and/or prevention of MS. Wagner et al. [126] (Acta Neuropathol. 2013 June; 125(6):795-813, hereby incorporated by reference) described Anle138b as an aggregation inhibitor for the therapy of neurodegenerative diseases such as prion disease. In one embodiment, Anle138b may therefore be useful for reducing and/or preventing the formation of PrP.sup.MS conformers.
[0098] In one embodiment, antibodies that selectively bind to PrP.sup.MS may be useful for reducing and/or preventing the formation of PrP.sup.MS conformers. In one embodiment, there is provided an antibody that selectively binds to PrP.sup.MS. The term antibody as used herein is intended to include monoclonal antibodies, polyclonal antibodies, antibody fragments, chimeric antibodies, single domain antibodies, humanized antibodies, human antibodies, single chain antibodies and bispecific antibodies. The antibody may be from recombinant sources and/or produced in transgenic animals. The term antibody fragment as used herein is intended to include Fab, Fab, F(ab)2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof and bispecific antibody fragments. Antibodies can be fragmented using conventional techniques. For example, F(ab)2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab)2 fragment can be treated to reduce disulfide bridges to produce Fab fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab and F(ab)2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.
[0099] In one embodiment, there is provided a method for the treatment or prevention of multiple sclerosis (MS) comprising administering to a subject an effective amount of agent that reduces and/or inhibits the formation of PrP.sup.MS conformers. Also provided is the use of an effective amount of agent that reduces and/or inhibits the formation of PrP.sup.MS conformers for the treatment or prevention of multiple sclerosis (MS) in a subject in need thereof. In one embodiment, the method further comprises identifying the subject as having, or at risk of development, MS as described herein, prior to administering to the subject an effective amount of agent that reduces and/or inhibits the formation of PrP.sup.MS conformers.
[0100] As used herein, the term effective amount refers to an amount of an agent that is sufficient to produce a desired effect, which can be a therapeutic, protective and/or beneficial effect. The effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent used or administered, the duration of the use or treatment, the nature of any concurrent treatments, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. As appropriate, an effective amount in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation. (See, for example, Remington, The Science and Practice of Pharmacy (20th ed. 2000)).
[0101] The terms treat, treating or treatment as used herein refer to any type of action that imparts a modulating effect, which, for example, can be a beneficial and/or therapeutic effect, to a subject afflicted with a condition, disorder, disease or illness, including, for example, improvement in the condition of the subject (e.g., in one or more symptoms), delay in the progression of the disorder, disease or illness, prevention or delay of the onset of the disease, disorder, or illness, and/or change in clinical parameters of the condition, disorder, disease or illness, etc., as would be well known in the art. The terms treat, treating or treatment as used herein also mean administering to a subject or the use of a therapeutically effective amount of the agents described herein and may consist of a single administration or use, or alternatively comprise a series of applications or uses.
[0102] As used herein, and as well understood in the art, treatment or treating is also an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment. Further any of the treatment methods or uses described herein can be formulated alone or for contemporaneous administration with other agents or therapies.
[0103] The terms prevent, preventing, and prevention and like terms are used herein to include imparting any level of prevention or protection which is of some benefit to a subject, such that there is a reduction in the incidence and/or the severity of the disease in a treated subject, regardless of whether the protection or reduction in incidence and/or severity is partial or complete.
[0104] For example, the treatment or prevention of MS may include alleviation or amelioration of one or more signs or symptoms of MS such as amyloid plaques, brain atrophy, demyelination, microgliosis, proliferation of oligodendrocyte precursors, ventriculomegaly and abnormal behavior. In one embodiment, the treatment or prevention of MS may include extending the time between relapses for a subject.
[0105] In one embodiment, the agents described herein may be for use, formulated for use, or administered to a subject in need thereof. In one embodiment, the agents described herein are formulated or modified to cross the blood brain barrier.
[0106] Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (200320th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.
[0107] Use or administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of use or administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple uses or administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.
[0108] Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray, drops or from an atomiser or dry powder delivery device); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly. In one embodiment, the route of administration is oral (e.g., by ingestion). In another embodiment, the route of administration is parenteral (e.g., by injection).
[0109] Also provided is a method for reducing and/or inhibiting the formation of PrP.sup.MS conformers in CNS cells or tissue. In one embodiment, the method comprises contacting CNS cells or tissue with an agent listed in Table 1. Optionally, the CNS cells or tissue are in vivo or in vitro. In one embodiment, the CNS tissue comprises cortical tissue, corpus callosum tissue, cerebellar tissue, optic nerve tissue and/or spinal dorsal column/dorsal root ganglion (DRG) tissue. In one embodiment, the CNS cells are brain cells or immune cells. In one embodiment, the brain cells are neurons, astrocytes, oligodendrocytes, oligodendrocyte precursor cells and/or microglia. In one embodiment, the immune cells are B cells and/or Follicular Dendritic cells.
[0110] The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples of certain embodiments of the invention.
EXAMPLES
Example 1: Detection of Prion Protein and Amyloid Deposits in MS
[0111] Using a conformationally-sensitive fluorescent amyloid probe (pFTAA) and spectral microscopy, widespread amyloid deposits were detected in cortex, white matter and around blood vessels in human MS brain (
[0112] Comparing the spectra of normal brain and mature amyloid from MS brain (
[0113] The background spectral shift in shown
Example 2: Investigations of PrP.SUP.MS .in Transgenic Mice Expressing Human PrP
[0114] Transmission experiments were performed using the tg650 mouse as the host, which overexpresses human PrP [68,69]. Six mice were injected intracerebrally with homogenate of human brain from a secondary progressive MS patient. A control group of tg650 mice was injected (n=5+2 uninjected age-matched) with non-MS human brain (terminal lung disease). The mice were followed by serial MRI brain imaging. After 1 year of incubation, MS-inoculated mice developed obvious brain atrophy and ventriculomegaly (
[0115] In addition, inoculated mouse brains frequently showed abnormal plaque-like PrP deposits. As shown in
[0116] Additional biochemical investigations into abnormal prion protein from tg650 mice were performed. Spin column fractionation revealed that MS-inoculated tg650 brain exhibited a shift to higher molecular weight complexes indicating an abnormally-aggregated PrP (
Example 3: Detection of MS Aggregated Protein in Leukocytes
[0117] Blood samples were obtained from 6 patients with MS and 5 non-MS controls including two patients with Alzheimer's disease. Leukocytes were isolated and stained with the fluorescent amyloid probe pentameric formyl thiophene acetic acid (pFTAA).
[0118] As shown in
[0119] The spectra of pFTAA in presence of MS PrP amyloid is specific to MS (
Example 4: Transmission to Mice Overexpressing Human PrP
[0120] Experiments are performed showing the primary inoculation from human brain to transgenic mouse, (termed 1st passage). Homogenates from frozen MS brain are prepared in sterile PBS and sonicated to break up larger aggregates (and any immune cells) to increase infectivity. 30 l of 1% homogenate is injected intracerebrally into 12 mice; in parallel, 100 l of 10% homogenate is injected intraperitoneally into 12 mice from each of 9 patients. Scrapie prions are known to be transmissible after oral ingestion and absorption from gut. Subtle myelopathy may develop in i.p. injected mice as the agent is transported into the cord from the peritoneal cavity. Controls from 12 non-MS patients are included to determine if detected changes are MS-specific.
[0121] Identical experiments are done using tga20 (murine prion overexpressor): it is predicted that human PrP.sup.MS will cross this species barrier with difficulty if at all. Mice may be incubated for extended periods to confirm this negative control. It is possible that tga20 may become infected, as the species barrier may not be absolute; this would not negate our hypothesis. Therefore the following controls are used: same experiments as above but into PrP null and wild type mice. Scrapie transmission depends on expression of endogenous prion protein and PrP nulls are resistant to infection [87]. Inoculation of wild type mice (expressing normal levels of murine prion) will exclude a non-specific pathological effect of injecting human brain into mouse. If human MS transmits to tg650 (human PrP overexpressor), but not to tga20 (mouse PrP overexpressor), this will provide very strong evidence for: a) prion as the pathogenic agent in MS, and b) specificity of PrP.sup.MS implying a unique human conformer. While selective transmission to human PrP-overexpressing mice, but not to WT with murine PrP or to PrP nulls, would provide strong evidence for a prion being the responsible agent, the possibility would still exist that a transmissible agent that was not a prion was responsible, and that the overexpressors were simply more susceptible to infection. To address this possibility, homogenates of human MS brain, and tg650 mouse brain successfully infected after 1st passage, will be immunodepleted of PrP using magnetic beads. No disease in mice injected with immunodepleted homogenate is expected, providing unequivocal evidence that prion protein was involved in the transmission. Equally important, these controls provide strong evidence against an unexpected infectious pathogen (e.g. a virus), or an adoptive immune transfer (e.g. activated T cells, antibodies), being responsible for transmission.
[0122] MRI: high-field MRI is used to non-invasively track development of pathology. Groups of animals (20 mice per month on average) are scanned using anatomical and quantitative T2 sequences. The former gives a complete z-stack through the entire brain, whereas the latter yields reliable data on pathological changes (mainly cellular edema/vacuolation/spongiform change) using our quantitative analysis techniques (
[0123] Passaging: A cardinal feature of prion diseases is shorter latency to disease and crossing of species barriers with repeated passaging, that is, harvesting previously inoculated brain after the first human-to-mouse injection and re-inoculating into nave mouse [88-90]. This intriguing phenomenon is thought to be due to rising titres and conformational adaption of the pathogenic prion to the host species [90,91]. Brain is harvested from significantly affected (by MRI) tg650 mice, and homogenates are injected i.c. and i.p. back into nave tg650 mice. Animals are followed by MRI, and then histology. A more rapid pathology is observed with 1st passage; this is repeated at least twice more in an attempt to accelerate pathology which eventually reaches a plateau [88]. Passaging provides further evidence for an adaptive prion as the pathogenic agent. Moreover if the latency to pathology can be reduced from 1 year to several months, this passaged brain material represents an invaluable reagent for creating a unique in vivo model of progressive MS.
[0124] Histology: Examination of fixed tissue with histology and immunochemistry is used to study pathological details typical of MS pathology. Techniques and probes include titrated digestion and amyloid probe staining to detect partially protease resistant, misfolded sheet-rich PrP aggregates. Examination of the spinal cord for demyelination, axonal injury and amyloid deposition after i.p. injection provides strong evidence that the pathogenic agent entered via afferents into the cord as is described in traditional scrapie [85], if cord pathology is observed in these and not in i.c. injected mice.
[0125] Biochemistry: Experiments are conducted to further characterize the PrP.sup.MS conformer. Brain homogenates from wild type, age-matched tg650 and inoculated tg650 (1.sup.st 2.sup.nd and higher passage) are digested with various concentrations of pronase E (0-30 g/ml) and run on Westerns, and probed with 6H4. Other PrP antibodies (3F4; 1E4 the latter being particularly good at detecting digested VPSPr's [93]) are also tested because cleavage at certain sites may variably affect immunoreactivity of the smaller remaining fragments. Passaging may increase the detectable titres of partially resistant cleavage products as is seen with scrapie [51,81,94]. The protocol is also performed on human MS brain samples to detect an abnormal PrP conformer. The experiments are repeated using thermolysin (0-150 g/ml), another protease that degrades PrPC while preserving both PK-sensitive and PK-resistant misfolded PrP isoforms [95,96].
[0126] Phosphotungstic acid (PTA) precipitation is very effective for eliminating normal un-aggregated proteins (including PrPC) and greatly improving detection of abnormally folded pathogenic PrP, regardless of protease sensitivity [92]. Control and inoculated tg650 mouse and human MS brain samples as above are precipitated using NaPTA ([92,97,98] and Fig. S1C). Additional pronase E digestion is tested to further isolate abnormal PrP. Pellets are resuspended, run on Westerns and probed with 6H4, 3F4 and 1 E4. This method should greatly increase ability to distinguish between normal PrPC vs. inoculated tg650 brain. Given the ability of PTA to concentrate and purify abnormal PrP by over 2 orders of magnitude while retaining its infectivity [92], these precipitates are also used for re-inoculation, which may greatly accelerate onset of pathology.
Example 5: Evidence of a MS Prionopathy in Human Material
[0127] Sections from MS brain and cord, from 6 SP and 6 PPMS patients are analyzed using hydrolytic and proteolytic techniques [106-108] to reveal abnormally folded/aggregated PrP over the normal PrPC background. Human MS and control brain samples are i) heated to 96 C. in citrate buffer, treated with formic acid (99%10min), both of which reduce PrPC staining, increasing contrast of abnormal PrP signal [46]. Exposure times are titrated to eliminate normal PrPC signal in control while maximizing abnormal PrP staining in MS brain. Preferred PrP antisera include 3F4 and 6H4 [109,110]. Titres are adjusted to minimize control, and maximize MS-related staining. Sections (either before or after heating/acid hydrolysis) are exposed to protease at varying concentrations.
[0128] Demonstration of partially protease-resistant PrP in MS brain, but not in controls, provides very strong evidence of a misfolded and likely pathogenic prion protein.
Example 6: MS Brain and Spinal Cord Transmits Pathology to Humanized Transgenic Mice
Materials and Methods
[0129] Human brain and spinal cord homogenates were prepared from controls (non-neurological death, Alzheimer's and Lewy body diseases, and chronic encephalitis) and from 10 primary and secondary progressive MS patients, sonicated and 30 l injected intracerebrally under anesthesia into tg650 human prion protein over-expressing mice (MM at codon 129). Animals were imaged using anatomical and quantitative T2 sequences on a Bruker 9.4T animal MRI. At termination, mouse brains were either frozen for biochemistry or fixed in formalin and paraffin embedded for histology and immunohistochemistry. Pathological PrP aggregates were immunodetected (3F4 antibody) after heat antigen retrieval and formic acid treatment to differentiate from PrP.sup.C. Immunofluorescence was performed using standard techniques. All error bars are s.e.m. P values by non-parametric Wilcoxon rank test.
Results
[0130] As shown in
[0131] In addition to the molecular pathologies observed in MS brain injected mice, injected mice exhibited abnormal behaviours not seen in controls. Specifically, MS brain-injected mice showed both deficits in a water maze test and as well as decreased exploratory behaviour/more anxiety (
[0132] As shown in
[0133] Quantitative T2 MRI analysis of 1.sup.st passage mice injected i.c. with MS brain homogenate demonstrated pathology in the corpus callosum and thalamus also seen in 2.sup.nd passage mice (
[0134] Human MS spinal cord homogenate was also demonstrated to result in the transmission of pathology. As shown in
Discussion
[0135] MS exhibits prominent features of both recurrent autoimmune inflammation and progressive degeneration. While current therapies are highly successful at mitigating the former, even potent immune suppression shows little benefit against progression. Together with reports of very early MS lesions showing profound demyelination with little inflammation, this suggests that MS may be a primary degenerative disorder, with the commonly seen inflammatory overlay an important, but secondary, phenomenon. The results presented herein are consistent with the hypothesis that MS, like other degenerative disorders, may be a transmissible protein misfolding disorder. One component might involve a disease-specific conformer of human prion protein. However, while seemingly transmissible across multiple passages, this conformer does not exhibit properties of more conventional transmissible spongiform encephalopathies, such as proteinase K resistance or abnormalities using conformationally-dependent immunoassay (data not shown).
[0136] Without being limited by theory, the MS-specific agent(s) may target the myelin and oligodendrocytes, possibly by chronically dysregulating the axo-myelinic synapse [120]. Chronic release of antigenic myelin debris, coupled with a dysregulated immune system, together may result in the characteristic inflammatory relapsing-remitting MS phenotype. In contrast, the same underlying degenerative process, but with a weaker autoimmune predilection, would instead promote a less inflammatory, more degenerative primary progressive course.
[0137] As set out above, human MS brain and spinal cord homogenates have been demonstrated to transmit pathology (MRI, histology, behavior) when injected either intracerebrally or intraperitoneally into human prion protein overexpressing mice. Remarkably, transmission of pathology persisted over multiple (4) passages. Histologically, MS-inoculated mouse brains exhibited FA resistant PrP deposits indicative of an abnormal conformer, typical of protein misfolding degenerations. The results presented herein are consistent with the hypothesis that an abnormal conformer of human prion protein (PrP.sup.MS) plays an important role in MS pathogenesis. Variable degrees of dysimmunity (determined by e.g. environment, genetics), convolved with a chronic cytodegeneration, can provide a unifying hypothesis to explain the broad spectrum of clinical MS phenotypes.
[0138] While the present disclosure has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the disclosure is not limited to the disclosed examples. To the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
[0139] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
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