Methods for determining abnormal CSF flow

Abstract

The present invention relates to biomarkers of cerebrospinal fluid (CSF) flow, methods for determining the synthesis rate and clearance rate of biomolecules in CSF of a subject, methods for determining abnormal CSF flow in a subject and diagnosing and treating disorders associated with abnormal CSF flow.

Description

METHODS FOR DETERMINING ABNORMAL CSF FLOWFIELD OF THE INVENTION

[0001] The present invention relates to biomarkers of cerebrospinal fluid (CSF) flow, methods for determining the synthesis rate and clearance rate of biomolecules in CSF of a subject, methods for determining abnormal CSF flow in a subject and diagnosing and treating disorders associated with abnormal CSF flow.BACKGROUND OF THE INVENTION

[0002] Normal pressure hydrocephalus (NPH) is a clinical syndrome characterised by excessive cerebrospinal fluid (CSF) accumulation resulting in ventriculomegaly, gait and cognitive impairment and urinary incontinence. Little is known about the disease mechanism(s) of NPH, but individuals often respond clinically to CSF diversion through lumbar or ventriculoperitoneal shunting. Mounting evidence from genetic and functional imaging studies supports that NPH is a disease characterized by abnormal CSF circulation. Two genetic risk factors have been identified; SFMBT1 and CFAP43, both implicating failure of CSF circulation.

[0003] Other disorders are associated with abnormal CSF flow include: other forms of hydrocephalus, idiopathic intracranial hypertension, Subarachnoid hemorrhage, Vestibular Schwanoma, CSF hypotension and Impaired CSF flow following intrathecal therapeutics.

[0004] The choroid plexus (ChP) has an important role in CSF production. It actively secretes and transports water, proteins and other metabolites across the blood-CSF barrier. It is unclear how CSF and its constituents are cleared from the subarachnoid space, but reabsorption can occur through the arachnoid granulations, and other pathways such as the meningeal lymphatics and the glymphatic system. It is not clear how excessive CSF arises in NPH, but ChP CSF hypersecretion, ependymal denudation, and damage and scarring of intraventricular and parenchymal (glia-lymphatic) CSF pathways have been postulated based on animal studies and observations in humans with acquired hydrocephalus (e.g., postinfection or hemorrhage).

[0005] To date, investigation of CSF circulation in vivo has been limited by a paucity of appropriate monitoring tools. Without in vivo biomarkers of CSF turnover, it has been challenging to accurately diagnose NPH and identify individuals likely to respond to shunt; to determine whether NPH is associated with dysregulation of CSF production, circulation, or clearance.

[0006] WO 2006 / 107814 relates to using Stable Isotope Labelling Kinetics (SILK) methods for measuring the metabolism of central nervous system derived biomolecules in a subject in vivo and to the diagnosis and treatment of neurological and neurodegenerative diseases, disorders, and associated processes.

[0007] The present invention addresses a need in the art for is a need for biomarkers of CSF flow and methods which can be used to determine CSF flow in subject.SUMMARY OF THE INVENTION

[0008] The present invention is defined in and by the appended claims.

[0009] The present invention provides a method for measuring the synthesis rate (SR) of a biomolecule in a test subject, comprising:(a) provision of a cerebrospinal fluid (CSF) sample obtained from the test subject, wherein the sample was taken at a first time period following exposure to a labeled amino acid under conditions which allowed incorporation of the labeled amino acid into the biomolecule;(b) detecting the amount of a biomolecule labeled with the labeled amino acid in the sample; and(c) either:a. calculating the synthesis rate (SR) from the ratio of labeled biomolecule in the CSF sample obtained from the test subject to a control sample, wherein the control sample is obtained from a healthy subject following exposure to the labeled amino acid under identical conditions to the test subject; or b. repeating step (a) at second time point and calculating the synthesis rate (SR) from the ratio of labeled biomolecule to unlabeled biomolecule determined by step (a) in the biological sample of a test subject over time.

[0010] The present invention also provides a method for measuring the clearance rate (CR) of a biomolecule in a test subject, comprising:(a) provision of a cerebrospinal fluid sample obtained from the test subject, wherein the sample was taken following exposure to a labeled amino acid under conditions which allowed incorporation of the labeled amino acid into the biomolecule during synthesis for a first time period followed by exposure to an unlabeled amino acid under conditions which allowed incorporation of the unlabeled amino acid into the biomolecule during synthesis for a second time period;(b) detecting the amount of the biomolecule labeled with the labeled amino acid and the amount of unlabeled biomolecule in a biological sample obtained from the test subject,(c) provision of one or more samples from the test subject at a later time period during which the test subject is only exposed to unlabeled amino acid and detecting the amount of biomolecule labeled with the labeled amino acid and the amount of the biomolecule not labeled with the amino acid in a biological sample obtained from the test subject; and(d) calculating the clearance rate (CR) from the ratio of labeled biomolecule to unlabeled biomolecule determined by step (b) and (c).

[0011] Suitably, in the methods of the invention, the biomolecule may be a choroid plexus(ChP) protein. Suitably, in the methods of the invention, the synthesis rate of one or more biomolecules may be determined. Suitably, in the methods of the invention, the one or more biomolecule(s) may be selected from the group consisting of: transthyretin (TThyrR), apolipoprotein E (APOE), cystatin C (Cys-C), serotransferrin, albumin, Serpin F1, FSTL 1, IGFBP2, Clusterin, GSTP1, ENPP2, TUBA1A, UCHL1, PEBP1, CFL1, PARK7, SOD1, or variants thereof. Suitably, in the methods of the invention, at least one biomolecule may be selected from the group consisting of: transthyretin (TThyrR), apolipoprotein E (APOE), cystatin C (Cys-C), serotransferrin and albumin.

[0012] Suitably, in the methods of the invention, the label may be a stable non-radioactive isotope. Suitably, in the methods of the invention, the label may be selected from13C or15N. Suitably, in the methods of the invention, the labeled amino acid may be13C Leucine,13C Arginine, or13C Lysine.

[0013] Suitably, in the method for measuring synthesis rate of a biomolecule, the sample(s) may be taken up to 28 hours following exposure to a labeled amino acid.

[0014] Suitably, in the methods for measuring clearance rate, the first sample may be taken from 0 to 72 hours following exposure to a labeled amino acid and one or more later samples are taken thereafter.

[0015] Suitably, the methods for determining synthesis rate and / or methods for determining clearance rate of the invention may further comprise:(a) comparing the SR and / or CR of a biomolecule in a test subject to a reference SR and / or CR representing a known disease or health status, and / or(b) comparing the SR and / or CR of a biomolecule in a test subject between two different time periods.

[0016] The present invention further provides a method of determining abnormal cerebrospinal fluid flow in a test subject and / or an increased risk of a developing a disorder associated with abnormal cerebrospinal fluid flow, the method comprising:(a) determining the synthesis rate and / or clearance rate of a biomolecule in the test subject in accordance with the methods of determining the synthesis rate and / or clearance rate of a biomolecule of the invention;(b) comparing the synthesis rate and / or clearance rate to a reference value or to the synthesis and / or clearance rate of a control healthy subject, wherein the process for obtaining the synthesis and / or clearance rate of the control healthy subject is identical to that of the test subject; and(c) determining abnormal cerebrospinal fluid flow and / or increased risk of a developing a disorder associated with abnormal cerebrospinal fluid flow in the test subject if the clearance rate and / or synthesis rate is lower than the reference value or at least 2-fold lower than the control healthy subject.

[0017] The present invention also provides a method of diagnosing a disorder associated with abnormal cerebrospinal fluid flow in a subject suspected of having a disorder associated with abnormal cerebrospinal fluid flow comprising:(a) determining the synthesis rate and / or clearance rate of a biomolecule in the subject in accordance with the methods of determining the synthesis rate and / or clearance rate of a biomolecule of the invention;(b) comparing the synthesis rate and / or clearance rate to a reference value or to the synthesis and / or clearance rate of a control healthy subject, wherein the process for obtaining the synthesis and / or clearance rate of the control healthy subject is identical to that of the subject suspected of having a disorder associated with abnormal cerebrospinal fluid flow; and (c) diagnosing a disorder associated with abnormal cerebrospinal fluid flow in the subject if the clearance rate and / or synthesis rate is lower than the reference value or at least 2-fold lower than the control healthy subject.

[0018] The present invention also provides the use of the methods of determining the synthesis rate and / or clearance rate of a biomolecule of the invention in diagnosing abnormal CSF flow in a test subject.

[0019] Suitably, the use may determine whether the subject has an increased risk of developing or diagnosing a disorder associated with abnormal CSF flow. Suitably, a disorder associated with abnormal CSF flow may be selected from the group consisting of: hydrocephalus, idiopathic intracranial hypertension, Subarachnoid hemorrhage, Vestibular Schwanoma, CSF hypotension and Impaired CSF flow following intrathecal therapeutics.

[0020] Suitably, the disorder may be hydrocephalus, optionally wherein the disorder may be normal pressure hydrocephalus.

[0021] The present invention further provides a method of treating a disorder associated with abnormal cerebrospinal fluid flow (CSF) in a subject identified as having abnormal cerebrospinal fluid flow in accordance with the invention or diagnosed as having a disorder associated with abnormal cerebrospinal fluid flow in accordance with the invention, wherein the method comprises performing lumbar or ventriculoperitoneal shunting.

[0022] The present invention also provides a method for measuring the synthesis rate (SR) of a biomolecule in vitro, comprising:(a) provision of an in vitro choroid plexus organoid fluid sample, wherein the sample was taken at a first time period following exposure to a labeled amino acid under conditions which allowed incorporation of the labeled amino acid into the biomolecule;(b) detecting the amount of a biomolecule labeled with the labeled amino acid in the sample; and(c) repeating steps (a) and (b) at second time point and calculating the synthesis rate (SR) from the ratio of labeled biomolecule to unlabeled biomolecule determined by step overtime.

[0023] The present invention further provides a method for measuring the clearance rate (CR) of a biomolecule in vitro, comprising:(a) provision of an in vitro choroid plexus organoid fluid sample, wherein the sample was taken following exposure to a labeled amino acid under conditions which allowed incorporation of the labeled amino acid into the biomolecule during synthesis; for a first time period followed by exposure to an unlabeled amino acid under conditions which allowed incorporation of the unlabeled amino acid into the biomolecule during synthesis for a second time period;(b) detecting the amount of a biomolecule labeled with the amino acid and a biomolecule not labeled with the labeled amino acid in a biological sample obtained from the test subject;(c) provision of one or more samples at a later time period during which the test subject is only exposed to unlabeled amino acid and detecting the amount of the biomolecule labeled with the amino acid and the biomolecule not labeled with the labelled amino acid in a biological sample obtained from the test subject; and(d) calculating the clearance rate (CR) from the ratio of labeled biomolecule to unlabeled biomolecule determined by step (b) and (c).

[0024] Suitably, in the in vitro methods of the invention, the biomolecule may be a choroid plexus (ChP) protein. Suitably, in the in vitro methods of the invention, the synthesis rate of one or more biomolecules may be determined. Suitably, in the in vitro methods of the invention, the one or more biomolecule(s) may be selected from the group consisting of: transthyretin (TThyrR), apolipoprotein E (APOE), cystatin C (Cys-C), serotransferrin, albumin, Serpin F1, FSTL 1, IGFBP2, Clusterin, GSTP1, ENPP2, TUBA1A, UCHL1, PEBP1, CFL1, PARK7, SOD1, or variants thereof. Suitably, in the in vitro methods of the invention, at least one biomolecule may be selected from the group consisting of: transthyretin (TThyrR), apolipoprotein E (APOE), cystatin C (Cys-C), serotransferrin and albumin.

[0025] Suitably, in the in vitro methods of the invention, the label may be a stable nonradioactive isotope. Suitably, in the in vitro methods of the invention, the label may be selected from 13C or 15N. Suitably, in the in vitro methods of the invention, the labeled amino acid is 13C Leucine, 13C Arginine, or 13C Lysine.

[0026] Suitably, in the in vitro methods for measuring synthesis rate of a biomolecule, the sample(s) may be taken up to 28 hours following exposure to a labeled amino acid.

[0027] Suitably, in the in vitro methods for measuring clearance rate, the first sample may be taken from 0 to 72 hours following exposure to a labeled amino acid and one or more later samples are taken thereafter.

[0028] The present invention further provides, use of a one or more proteins selected fromthe group consisting of transthyretin (TThyrR), apolipoprotein E (APOE), cystatin C (Cys-C), serotransferrin, albumin, Serpin F1, FSTL 1, IGFBP2, Clusterin, GSTP1, ENPP2, TLIBA1A, LICHL1, PEBP1, CFL1, PARK7, SOD1, or variants thereof as a biomarker for: determining cerebrospinal fluid flow, identifying as subject at risk of developing a disorder associated with abnormal cerebrospinal fluid flow, or diagnosing a disorder associated with cerebrospinal fluid flow.

[0029] Suitably, the disorder may be selected from the group consisting of: hydrocephalus, idiopathic intracranial hypertension, Subarachnoid hemorrhage, Vestibular Schwanoma, CSF hypotension and Impaired CSF flow following intrathecal therapeutics.

[0030] Suitably, the disorder may be hydrocephalus, optionally wherein the disorder may be normal pressure hydrocephalus.

[0031] These and other aspects are addressed in more detail in the description set forth below.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The invention will further be illustrated by reference to the following figures:

[0033] Fig.1 shows choroid plexus organoid characteristics and protein synthesis rates.

[0034] Fig.1a shows a bright field image of a H1 ChP organoid at day 42 that developed self-contained fluid compartments with CSF-like fluid.

[0035] Fig.1b shows a representative confocal image of whole ChP organoid from H1 at day 40 stained forTTHY in green and DAPI in blue.

[0036] Fig.1c shows a representative confocal image of ChP epithelium from a H9 derived organoid at day 60 stained for ChP marker MSXIand CSF transporter NKCC1, nuclei are stained with DAPI (blue).

[0037] Fig.ld shows a bar chart with scatter plots of mass spectrometry detected emPAI values for ALB peptides of human and bovine origin from organoid media, organoid CSF from 3 independent cell lines, i.e. human embryonic lines H9, H1 and iPSC line IMR90-4, bovine foetal CSF and human adult CSF (mass spectrometry data from Pellegrini et al.).

[0038] Fig.le relates to single cell RNA (scRNA) sequencing of ChP organoids (ChP day 27, ChP day 46, ChP day 53) and cortical organoid (control day 55) showing the different cell populations: ChP stroma, neurons, ChP epithelial cells (Mat ChP), hem / ChP progenitors (Imm ChP / hem) and neuronal progenitors (Neural prog).

[0039] Fig.1f relates to scRNA sequencing showing different timepoints analysed, i.e. ChP day 27, ChP day 46, ChP day 53, Ctrl day 55.

[0040] Fig.1g relates to a dot plot showing average expression and percentage of cells expressing the displayed enriched genes identified by scRNA sequencing (scRNA seq datafrom Pellegrini et al.). ALB, albumin; ChP, choroid plexus; CSF, cerebrospinal fluid; NKCC1, Na+ / K+ / 2CI- cotransporter; NPH, normal pressure hydrocephalus; TTHY, transthyretin.

[0041] Fig.lh shows a bar chart comparing FSR% (Iog10 transformed) choroid plexus proteins synthesized by organoid (blue) labelled by SILK, and human controls labelled by SILK (green); FSR: fractional synthesis rate. Fig.2 relates to choroid plexus protein turnover in human CSF: plasma and choroid plexus derived proteins.

[0042] Fig.2a shows a schematic of SILK labelling method in vivo.

[0043] Fig.2b shows an in vivo SILK time course profile of albumin peptide in both normal pressure hydrocephalus and control CSF.

[0044] Fig.2c shows an in vivo SILK time course profile of serotransferrin peptide in both normal pressure hydrocephalus and control CSF.

[0045] Fig.2d shows an in vivo SILK time course profile of transthyretin peptide in both normal pressure hydrocephalus and control CSF.

[0046] Fig.2e shows an in vivo SILK time course profile of cystatin C peptide in both normal pressure hydrocephalus and control CSF.

[0047] Fig.2f shows an in vivo SILK time course profile of apolipoprotein E peptide in both normal pressure hydrocephalus and control CSF.

[0048] Fig.3 provides a summary of choroid plexus protein (ChP) kinetics in vivo.

[0049] Fig.3a shows a kinetic curve of five peptides within a single subject (NPH).

[0050] Fig.3b shows a kinetic curve of five peptides within a single subject (control)

[0051] Fig.3c shows a comparison of ChP kinetics in ventricular and lumbar CSF

[0052] Fig.3d shows a correlation of fractional synthesis rate of TTHY peptides ALG (p=0.037, p=0.738, R2=0.483) and TSE (p=0.042, p=0.683, R2=0.519) with CSF production rate by LiquoGuard.

[0053] Fig.3e shows the TTHY turnover in NPH patients (n=8), controls (n=4) and ChP organoids (n=2).

[0054] Fig.4 shows NPH imaging.

[0055] Fig.4a shows VCSF segmentation outputs obtained using deep-learning-derived CT brain quantification pipeline in two representative pre- and post-shunt paired CT datasets.

[0056] Fig.4b shows VCSF volume changes (in %) between pre- and post-shunt VCSF images.

[0057] Fig.4c shows VCSF volumes in millilitres before and after shunt.DETAILED DESCRIPTION OF THE INVENTION

[0058] The present invention will now be described with reference to the accompanying drawings, in which representative embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0060] The terms "treatment" and "treating" herein refer to an approach for obtaining beneficial or desired results in a subject, which includes a therapeutic benefit.

[0061] “Therapeutic benefit” refers to eradication, amelioration or slowing the progression of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the patient may still be afflicted with the underlying disorder.

[0062] The term "subject" refers to any suitable subject, including any animal, such as a mammal. In preferred embodiments described herein, the subject is a human.

[0063] The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") includes those embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, that "consist of’ or "consist essentially of’ the described features. The term “comprises” or “comprising” can be used interchangeably with “includes”.

[0064] Unless context explicitly states otherwise, it is envisaged that any embodiment described herein may be combined with any other embodiment described herein. Similarly, the features of any dependent claim (i.e., representing preferred embodiments of the present invention) may be readily combined with the features of any of the independent claims or other dependent claim or embodiments, unless context clearly dictates otherwise.

[0065] When ranges are used herein, all combinations and sub-combinations of ranges and specific embodiments therein are intended to be included. The term "about" orwhen referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary. Typical experimental variabilities may stem from, for example, changes and adjustments necessary during scale-up from laboratory experimental and manufacturing settings to large scale. When referring to time “about” is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

[0066] It must be noted that as used herein and in the appended claims, the singular forms"a", "an", and “the” include plural referents unless the context clearly dictates otherwise.

[0067] Also as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0068] “Clearance rate” refers to the rate at which the biomolecule of interest is removed.

[0069] “Fractional clearance rate” or FCR is calculated as the natural log of the ratio of labeled biomolecule over a specified period of time.

[0070] “Synthesis rate” refers to the rate at which the biomolecule of interest is synthesized.

[0071] “Fractional synthesis rate” or FSR is calculated as the slope of the increasing ratio of labeled biomolecule over a specified period of time divided by the predicted steady state value of the labeled biomolecule.

[0072] “Lag time” generally refers to the delay of time from when the biomolecule is first labeled until the labeled biomolecule is detected.

[0073] The “synthesis window” generally refers to the window of time after the large phase until rate of incorporation of the labelled amino acid in the biomolecule plateaus. The skilled person readily understands that the methods and uses of the invention utilize one or more CSF samples obtained during the synthesis window to calculate the synthesis rate or to use the synthesis rate of the biomolecule to: determine abnormal CSF, identify a subject at risk of a disorder associated with abnormal CSF flow or to diagnose a disorder associated with abnormal CSF flow.

[0074] The “clearance window” generally refers to the window of time when a reduction in labelled biomolecule is first detected until the labelled biomolecule is no longer detected.Biomolecule

[0075] The term “biomolecule” refers to a protein present in the cerebrospinal fluid of a subject that may be used as a biomarker of cerebrospinal fluid turnover.

[0076] The present inventors have surprisingly found that proteins abundantly expressed by the choroid plexus (ChP) may have utility as biomarkers of choroid plexus function and illustrated thorough a panel of choroid plexus derived proteins that such proteins can be used to determine CSF flow in a subject. These biomolecules were characterized and used to measure synthesis and turnover rates in human stem cell-derived ChP organoids and in human CSF. Patients with suspected normal pressure hydrocephalus were compared to patients that had recovered from non-traumatic subrachoid hemorrhage such that CSF flow rate had plateaued to within normal clinical parameters as controls and the protein kinetics of biomolecules of the invention have been validated as biomarkers of CSF flow showing significant correlation with CSF flow as measured in-clinic.

[0077] Accordingly, the biomolecule used in the methods and uses described herein may be a choroid plexus (ChP) protein.

[0078] Furthermore, the present inventors have surprisingly found that biomolecules such as albumin and serotransferrin may also be used as biomarkers of abnormal CSF flow. These biomolecules are more likely to be synthesized by the liver and then transported across the blood-CSF barrier allowing the synthesis and clearance rates of such biomolecules to be calculated from CSF samples.

[0079] Suitably, the biomolecule may be selected from the group consisting of: transthyretin (TThyrR), apolipoprotein E (APOE), cystatin C (Cys-C), serotransferrin, albumin, Serpin F1, FSTL 1, IGFBP2, Clusterin, GSTP1, ENPP2, TUBA1A, UCHL1, PEBP1, CFL1, PARK7, SOD1, or variants thereof. Suitably, the biomolecule may be selected from the group consisting of: transthyretin (TThyrR), apolipoprotein E (APOE), cystatin C (Cys-C), serotransferrin and albumin.

[0080] Suitably, the protein sequence for transthyretin may comprise ALGISPFHEHAEVVFTANDSGPR (SEQ ID NO: 1) or TSESGELHGLTTEEEFVEGIYK (SEQ ID NO: 2).

[0081] Suitably, the protein sequence for cystatin C may comprise ALDFAVGEYNK (SEQ ID NO: 3).

[0082] Suitably, the protein sequence for apolipoprotein E may comprise LQAEAFQAR (SEQ ID NO: 4).

[0083] Suitably, the protein sequence for serotransferrin may comprise MYLGYEYVTAIR (SEQ ID NO: 5).

[0084] Suitably, the protein sequence for albumin may comprise TYETTLEK (SEQ ID NO: 6).

[0085] In the methods of the invention the synthesis rate or clearance rate is determined of at least one or more biomolecules selected from the group consisting of: transthyretin (TThyrR), apolipoprotein E (APOE), cystatin C (Cys-C), serotransferrin, albumin, Serpin F1, FSTL 1, IGFBP2, Clusterin, GSTP1, ENPP2, TUBA1A, UCHL1, PEBP1, CFL1, PARK7, SOD1, or variants thereof. Suitably, the biomolecule may be selected from the group consisting of: transthyretin (TThyrR), apolipoprotein E (APOE), cystatin C (Cys-C), serotransferrin and albumin may be measured. In such methods the synthesis rate or clearance rate of a panel of biomolecules may be measured. The panel may comprise at least one, at least two, at least three, at least four or at least five biomolecules selected from the group consisting of: transthyretin (TThyrR), apolipoprotein E (APOE), cystatin C (Cys-C), serotransferrin, albumin, Serpin F1, FSTL 1, IGFBP2, Clusterin, GSTP1, ENPP2, TUBA1A, UCHL1, PEBP1, CFL1, PARK7, SOD1, or variants thereof. Suitably, the biomolecule may be selected from the group consisting of: transthyretin (TThyrR), apolipoprotein E (APOE), cystatin C (Cys-C), serotransferrin and albumin, optionally in combination with other proteins.

[0086] As used herein “unlabeled biomolecule” refers to a biomolecule in which a labelledamino has not been incorporated during synthesis of the biomolecule.

[0087] As used herein “labelled biomolecule” refers to a biomolecule in which a labelled amino acid has been incorporated during synthesis of the biomolecule.

[0088] In the methods and uses of the invention, CSF samples are provided at one or more time points from which the rate of synthesis or rate of clearance of the biomolecule can be determined. Such timepoints are determined with regard to the amount of labelled amino acid administered to the subject and the expected synthesis or clearance window of the biomolecule. Suitably, multiple CSF samples may be taken at multiple timepoints. For example, where two samples are taken, they may be taken at a first timepoint and a second timepoint, where three samples are taken they may be taken at a first timepoint, second timepoint and third timepoint etc. Suitably, a plurality of samples are taken such as at least 2 or at least 3 or at least 4 or at least 5 or at least 6 or at least 7 or at least 10. Where either synthesis or clearances rates for a biomolecule of interest are not known, multiple samples may be taken at regular time points up to 72 hours after exposure, though for methods of determining synthesis rates of a biomolecule timepoints up to 28 hours will typically suffice.

[0089] Without wishing to be bound by theory, suitable timepoints for determining clearance rates of albumin and serotransferrin may be up to 48 hours to 10 days, preferably up to 3 days; suitable timepoints for determining clearance rates of transthyretin and cystatin C may be up to 20 hours to 48 hours, preferably up to 36 hours; and suitable timepoints for determining clearance rates of apolipoprotein may be up to 20 hours to 5 days, preferably up to 3 days.

[0090] Suitably, once the typical synthesis and clearance windows are known a single timepoint may be used to determine abnormal CSF flow by comparison with a known reference value (such as a reference value for normal clinical CSF at the same timepoint).Labelled amino acid

[0091] Suitably, the labelled amino acid may be an amino acid which is labelled with an isotope. The biomolecules used in the methods of the invention may be peptides. Any labelled amino acid may be used which is capable of being incorporated into the peptide sequence of the biomolecule during synthesis. Accordingly, labelled amino acids may be picked in accordance with the peptide sequence of a biomolecule.

[0092] A person of ordinary skill in the art is readily aware of different moieties which may be used to label the amino acid. For example, a non-radioactive isotope may be utilized and the presence of a non-radioactive isotope in a labelled biomolecule could be measured by mass spectroscopy. For example, the labeled amino acid may be an amino acid comprising a non-radioactive isotope (such as13C).

[0093] Considerations when choosing which labelled amino acid to use in the methods and uses of the invention include:• The amino acid being present in at least one residue of the biomolecule into which is it is to be incorporated;• The suitability of the amino acid to quickly reach the site of protein synthesis and rapidly equilibrate across the blood-brain barrier;• The applicability of using an essential amino acid to increase the percentage labelled being incorporated into the biomolecule;• Availability and validation of the labelled amino acid to be used, such as for in vivo labelling of a subject.

[0094] In view of the above considerations, preferably the labelled amino acid may be.13C Leucine,13C Arginine, or13C Lysine. Suitably,13Ce leucine may be used.

[0095] In the methods and uses of the invention, a sample is provided of a CSF. Prior to the provision of a CSF sample, the subject is exposed to a labelled amino acid under conditions which allow the labelled amino acid to be incorporated into the biomolecule as it is synthesized. This may be achieved by administering a labelled amino acid to the subject by any suitable means. Suitably, this may be intravenously.

[0096] The dose of labelled amino acid is selected to enable ratios of labelled to unlabeled biomolecules to be determined from the CSF sample at the timepoint taken. For example, 1 to 4 mg / kg / hr of labeled amino acid may be administered intravenously for 1 to 15 hours. Suitably,13C labelled amino acid may be administered intravenously at a rate to 2 to 3 mg / kg / hr for up to 10 hours.

[0097] Alternatively, in some embodiments, the methods and uses of the invention the biomolecule may instead be labeled using a nucleoside triphosphate comprising a nonradioactive isotope (such as15N). In such embodiments, in the methods and uses of the invention, the term “amino acid” or “labelled nucleic acid” may be replaced with “nucleoside triphosphate” or “labelled nucleoside triphosphate”.Method for measuring the synthesis rate (SR) of a biomolecule in a test subject

[0098] The present invention provides a method for measuring the synthesis rate (SR) of a biomolecule in a test subject, comprising:(a) provision of a cerebrospinal fluid (CSF) sample obtained from the test subject, wherein the sample was taken at a first time period following exposure to a labeled amino acid under conditions which allowed incorporation of the labeled amino acid into the biomolecule;(b) detecting the amount of a biomolecule labeled with the labeled amino acid in the sample; and(d) either:a. calculating the synthesis rate (SR) from the ratio of labeled biomolecule in theCSF sample obtained from the test subject to a control sample, wherein the control sample is obtained from a healthy subject following exposure to the labeled amino acid under identical conditions to the test subject; or b. repeating step (a) at second time point and calculating the synthesis rate (SR) from the ratio of labeled biomolecule to unlabeled biomolecule determined by step (a) in the biological sample of a test subject over time.

[0099] Suitably, the method may encompass repeating step (a) multiple times, each at a subsequent timepoint (e.g., a third timepoint, fourth timepoint, fifth timepoint etc.) compared to the previous timepoint. Calculation of the synthesis rate (SR) can be made by comparison of the ratio of labeled biomolecule to unlabeled biomolecule determined at two or more of the timepoints.

[0100] The synthesis rate can be measured from one or more samples of the test subject. The synthesis of the biomolecule may be calculated by labeled / unlabeled biomolecule ratio in the sample to that of a healthy control taken at the same timepoint as the test subject’s sample, where the control followed exposure to the same labelled amino acid under identical conditions to the test subject. Alternatively, the synthesis rate could be measured based on a comparison to a predetermined curve of labeled / unlabeled biomolecule ratio of the molecule over time. Such comparison would enable a synthesis rate to be determined that may be associated with abnormal CSF flow. For example, if the synthesis rate were 2 or more-fold lower compared to a control or predetermined curve for normal CSF flow.

[0101] The synthesis rate may be measured using a slope, the exponential fit curve, or a compartmental model fit that defines the rate of synthesis for the biomolecule under the conditions used. For such calculations, a minimum of one sample is typically required (e.g., one could estimate the baseline label), two are preferred, and multiple samples are more preferred to calculate the ratio of labeled to unlabeled protein overtime. Multiple samples may provide a more accurate curve of the uptake of the label into the biomolecule (i.e., the synthesis rate). The amount of labeled protein in a biological sample at a given time reflects the synthesis rate and may be expressed as percent per hour or the mass / time (e.g., mg / hr).

[0102] Such methodology is illustrated in the examples, the in vivo synthesis and clearance rate may be measured by administering labeled13Ce leucine intravenously at a rate of 3mg / kg / hr for 10 minutes, followed by 2mg / kg / hrfor up to 9 hours and collecting CSF samples at regular intervals. The amount of labeled and unlabeled biomolecule in the biological samples is typically determined by immunoprecipitation followed by LC-ESI-tandem MS.

[0103] A person of ordinary skill in the art is readily aware that the synthesis rate may be determined by various methods such as by mol faction label or by fractional synthesis rate as detailed in Peterson et al., “SILK studies - capturing the turnover of proteins linked toneurodegenerative diseases”, Nature Reviews Neurology, 15, 419-427 (2019) incorporated herein by reference.

[0104] Suitably, the methodology may allow measurement of the labeled and unlabeled biomolecule (such as a choroid peptide) at the same time, so that the ratio of labeled to unlabeled protein, as well as other calculations, may be made.

[0105] Suitably, the method of measuring the synthesis rate may be by determining the fractional synthesis rate (FSR). The FSR equals the initial rate of increase of labeled to unlabeled protein divided by the precursor enrichment.

[0106] Other parameters, such as lag time and isotopic tracer steady state, may be determined and used as measurements of the biomolecule's metabolism and physiology.

[0107] The preferred timepoints post exposure to the labelled amino acid will depend on the biomolecule to be measured.Method for measuring the clearance rate (CR) of a biomolecule in a test subject

[0108] The present invention also provides a method for measuring the clearance rate (CR) of a biomolecule in a test subject, comprising:(a) provision of a cerebrospinal fluid sample obtained from the test subject, wherein the sample was taken following exposure to a labeled amino acid under conditions which allowed incorporation of the labeled amino acid into the biomolecule during synthesis for a first time period followed by exposure to an unlabeled amino acid under conditions which allowed incorporation of the unlabeled amino acid into the biomolecule during synthesis for a second time period;(b) detecting the amount of the biomolecule labeled with the labelled amino acid and the amount of unlabeled biomolecule in a biological sample obtained from the test subject,(c) provision of one or more samples from the test subject at a later time period during which the test subject is only exposed to unlabeled amino acid and detecting the amount of biomolecule labeled with the labeled amino acid and the amount of the biomolecule not labeled with the amino acid in a biological sample obtained from the test subject; and(d) calculating the clearance rate (CR) from the ratio of labeled biomolecule to unlabeled biomolecule determined by step (b) and (c).

[0109] After the administration of labeled amino acid is terminated, the rate of decrease of the ratio of labeled to unlabeled protein may reflect the clearance rate of that protein. For these calculations, a minimum of one sample is typically required (one could estimate the baseline label), two are preferred, and multiple samples are more preferred to calculate an accurate curve of the decrease of the label from the protein over time (i.e. , the clearance rate). The amount of labeled protein in a biological sample at a given time during the clearance window can reflect the clearance rate (i.e., removal or destruction) and is usually expressed as percentper hour or the mass / time (e.g., mg / hr) of the protein in the subject.

[0110] Suitably, in the methods for measuring clearance rate, the first sample may be taken from 0 to 72 hours following exposure to a labeled amino acid and one or more later samples are taken thereafter.

[0111] Suitably, the methods for determining clearance rate of the invention may further comprise:(a) comparing the clearance rate of a biomolecule in a test subject to a reference clearance rate representing a known disease or health status, and / or(b) comparing the clearance rate of a biomolecule in a test subject between two different time periods.

[0112] Where the clearance rate is determined utilizing two more or more CSF samples taken at different time points, suitably the timepoints may be up to 5 hours apart, preferably up to 4 hours apart, preferably up to 3 hours apart. The samples may be taken at regular timepoints so that the samples are taken for e.g., 15 minutes or 30 minutes or an hour apart.

[0113] A person of ordinary skill in the art is readily aware that samples are provided at timepoints chosen to be within the clearance window of the biomolecule. For example, time points may be selected having regard to the clearance window of the selected biomolecule in a subject having healthy CSF flow.

[0114] When the clearance window of a biomolecule is not known, multiple samples can be taken at regular timepoints and two or more samples within the clearance window may be chosen to calculate the clearance rate.Methods for: determining abnormal CSF flow, identifying subjects with an increased risk of developing a disorder associated with abnormal CSF flow and diagnosing a disorder associated with CSF flow

[0115] A number of disorders are associated with abnormal cell flow. Such disorders include: hydrocephalus, idiopathic intracranial hypertension, Subarachnoid hemorrhage, Vestibular Schwanoma, CSF hypotension and Impaired CSF flow following intrathecal therapeutics.

[0116] A subject may be considered to have “abnormal CSF flow” if the clearance rate and / or synthesis rate of a biomolecule of the invention has at least a two-fold change compared to the normal clearance rate parameters and / or synthesis rate parameters associated with normal CSF flow.

[0117] The normal synthesis and / or clearance rate parameters for a particular biomolecular biomarker may be determined using a healthy control such as a subject identified as having normal CFS using the LiguoGuard clinical protocol.

[0118] Current methodology for determining CSF is both invasive, painful, and costly.Prolonged diagnostic lumbar drainage is currently the gold standard diagnostic test to identify individuals likely to clinically respond to CSF diversion - see Grunewald RA. Normal pressure hydrocephalus. BMJ Best Practice. Accessed August 22, 442 2024. https: / / bestpractice.bmj.com / topics / en-gb / 712. However, this method requires -three day admission to hospital. A lumbar drain is inserted between the L3 / L4 spinal vertebrae and LiquoGuard external drainage tubing is attached to the drainage catheters. The external intracranial pressure (ICP) transducer of the LiquoGuard pump is applied on the body of the patients in line with the external auditory meatus. Participants are requested to remain lying flat to allow CSF flow rates to stabilize and for the calculation of the CSF production rate. As such, less invasive methods to determine abnormal CSF flow are needed.

[0119] The present invention provides methods of determining abnormal CSF flow which are less invasive, more cost-effective, and easier to administer.

[0120] As shown in the examples, various biomolecules have been identified which can be used as a biomarker for CSF flow. Suitable biomolecules may be selected from the group consisting of: transthyretin (TThyrR), apolipoprotein E (APOE), cystatin C (Cys-C), serotransferrin, albumin, Serpin F1, FSTL 1, IGFBP2, Clusterin, GSTP1, ENPP2, TLIBA1A, LICHL1, PEBP1, CFL1, PARK7, SOD1, or variants thereof. Suitably, the biomolecule may be selected from the group consisting of: transthyretin (TThyrR), apolipoprotein E (APOE), cystatin C (Cys-C), serotransferrin and albumin.

[0121] A panel of biomolecules may be measured of at least one, at least two, at least three, at least four or at least five biomolecules selected from the group consisting of: transthyretin (TThyrR), apolipoprotein E (APOE), cystatin C (Cys-C), serotransferrin, albumin, Serpin F1, FSTL 1, IGFBP2, Clusterin, GSTP1, ENPP2, TUBA1A, UCHL1, PEBP1, CFL1, PARK7, SOD1, or variants thereof. Suitably, the biomolecule may be selected from the group consisting of: transthyretin (TThyrR), apolipoprotein E (APOE), cystatin C (Cys-C), serotransferrin and albumin, optionally in combination with other proteins.

[0122] The Examples have demonstrated that CSF flow may be correlated to the clearance rate and / or synthesis rate of the biomolecule.

[0123] Accordingly, the present invention provides a method of determining cerebrospinal fluid flow in a test subject and / or an increased risk of a developing a disorder associated with abnormal cerebrospinal fluid flow, the method comprising:(a) determining the synthesis rate and / or clearance rate of a biomolecule in the test subject in accordance with the methods of determining the synthesis rate and / or clearance rate of a biomolecule of the invention;(b) comparing the synthesis rate and / or clearance rate to a reference value or to the synthesis and / or clearance rate of a control healthy subject, wherein the process for obtainingthe synthesis and / or clearance rate of the control healthy subject is identical to that of the test subject; and(c) determining (i) cerebrospinal fluid flow and / or (ii) increased risk of a developing a disorder associated with abnormal cerebrospinal fluid flow in the test subject if the clearance rate and / or synthesis rate is lower than the reference value or at least 2-fold lower than the control healthy subject.

[0124] The present invention also provides a method of diagnosing a disorder associated with abnormal cerebrospinal fluid flow in a subject suspected of having a disorder associated with abnormal cerebrospinal fluid flow comprising:(a) determining the synthesis rate and / or clearance rate of a biomolecule in the subject in accordance with the methods of determining the synthesis rate and / or clearance rate of a biomolecule of the invention;(b) comparing the synthesis rate and / or clearance rate to a reference value or to the synthesis and / or clearance rate of a control healthy subject, wherein the process for obtaining the synthesis and / or clearance rate of the control healthy subject is identical to that of the subject suspected of having a disorder associated with abnormal cerebrospinal fluid flow; and (c) diagnosing a disorder associated with abnormal cerebrospinal fluid flow in the subject if the clearance rate and / or synthesis rate is lower than the reference value or at least 2-fold lower than the control healthy subject.

[0125] Two genetic risk factors have been identified; SFMBT1 and CFAP43, both implicating failure of CSF circulation. Accordingly, a subject may be suspected of having a disorder associated with abnormal cerebrospinal fluid flow if the subject has either or both of these genetic risk factors.

[0126] Suitably, a subject may be considered as being at increased risk of a disorder associated with abnormal flow or diagnosed with a disorder associated with abnormal CSF flow when there is at least a 2-fold change, or at least a 3-fold change, or at least 4-fold change, or at least a 5-fold change, or at least a 6-fold change or at least a 7-fold change or at least an 8-fold change or at least a 9-fold change or at least a 10-fold change compared to a predetermined rate associated with normal CSF flow.

[0127] For example, the present inventors have demonstrated that the clearance rate of transthyretin is approximately 10 times lower in subjects diagnosed with normal pressure hydrocephalus (NPH). A similar trend was seen for cystatin C. Hence, the present invention provides methods of determining if a subject is at increased risk of developing hydrocephalus (such as NPH) and diagnosing hydrocephalus (such as NPH).

[0128] Suitably, a subject may be considered as being at increased risk of a disorder associated with abnormal flow if they have symptoms associated with a disease associated with abnormal CSF. BMJ best practices may be utilized to identify a subject as being atincreased of a disorder associated with abnormal flow. For example, the subject may have symptoms identified in BMJ best practices as associated with one of hydrocephalus, idiopathic intracranial hypertension, Subarachnoid hemorrhage, Vestibular Schwanoma, CSF hypotension and Impaired CSF flow following intrathecal therapeutics. Suitably, a subject may be diagnosed as having a disorder associated with abnormal CSF from the identification of abnormal CSF by the methods of the invention in combination with one or more key diagnostic indicators, other diagnostic indicators and / or risk factors outlined in BMJ best practices for the specific disorder associated with abnormal CSF flow.Kits for determining abnormal CSF flow.

[0129] The present invention also provides kits for diagnosing or monitoring abnormal CSF flow in a subject by measuring the clearance rate and / or synthesis rate of a biomolecule according to the invention. Generally, a kit comprises a labeled amino acid, means for administering the labeled amino acid, means for collecting biological samples overtime, and instructions for detecting and determining the ratio of labeled to unlabeled biomolecule so that a clearance and / or synthesis rate may be calculated. The synthesis and / or clearance rate may then be compared to the clearance rate and / or synthesis rate of a normal, healthy individual or compared to the clearance rate and / or synthesis rate from the same subject generated at an earlier time. These comparisons may enable a practitioner to predict the advent of abnormal CSF flow, or monitor the progression of abnormal CSF flow, or verify the effectiveness of a treatment for abnormal CSF flow. In a preferred embodiment, the kit comprises13Ce -leucine and instructions how to determine the rate of clearance and / or synthesis of one or more of transthyretin (TThyrR), apolipoprotein E (APOE), cystatin C (Cys-C), serotransferrin, albumin, Serpin F1, FSTL 1, IGFBP2, Clusterin, GSTP1, ENPP2, TUBA1A, UCHL1, PEBP1, CFL1, PARK7, SOD1, or variants thereof.ExamplesMaterials and MethodsStable Isotope Labelling Kinetics (SILK) in ChP organoidsGeneration of ChP organoids

[0130] Human H1 ES cells were obtained from WiCell and used for the generation of choroid plexus organoids using Stem Cell Technologies Cerebral Organoid kit (catalog nos. 08570 and 08571) as previously described in Pellegrini L, Bonfio C, Chadwick J, Begum F, Skehel M, Lancaster MA. Human CNS barrier forming organoids with cerebrospinal fluid production.Science. 2020; 369(6500) :eaaz5626. Specifically, EBs were generated by seeding 4000 cells in a 96-well II bottom low attachment plate with EB media and 50 pM Y-27632 ROCK inhibitor for 3 days. On day 5, the culture media was switched to Nl (neural induction) withing the same 96-well plate. On day 7, EBs were embedded in 30 pl of Matrigel (Corning) using dimpled parafilm sheets, following the established procedure described by Lancaster et al.1, and incubated for 20min at 37°C. Subsequently, the EBs were transferred to a 6-well plate, with each well containing 3ml of Expansion media. For ChP patterning, 3 pM CHIR and 20 ng / ml BMP4 were added in Maturation media on day 10 until day 17. Starting from day 30, dissolved Matrigel (at a ratio of 1 :50) was introduced to the Maturation media.ChP profiling by single cell RNA sequencing

[0131] Organoid single-cell dissociation, library preparation and sequencing were all previously described in Leinonen V, Koivisto AM, Savolainen S, et al. Raw and processed human organoid sequencing data are available at the Gene Expression Omnibus (GEO) database GSE150903. Specifically, Single-cell dissociation was performed by first pooling two organoids for each condition: 55-day H9 telencephalic organoids, 27-day H1 ChP (ChP sample 1), 46-day H1 ChP (ChP sample 2), and 53-day H1 ChP (ChP sample 3) into a 15 mL tube. Samples were incubated in 1ml of Accumax (Sigma, A7089) with 400 pg DNase I and 15 pM actinomycin D at 37°C for 20min with gentle agitation. At 5min intervals, the sample tubes were flicked then pipetted 10 times. Clumps were allowed to settle and supernatant collected, to which 100 pL FBS was added before filtration on a 35pm filter tube (Corning, 352235). Samples were then spun at 300g for 5min. Dead Cell Removal kit and MACS column (Miltenyi, 130- 090-101) were used to remove dead cells before another spin at 300g for5min. Cells were resuspended in an appropriate volume of 0.04% BSA in PBS to load 16,000 cells per well on the 10X Chromium system (10X Genomics).ChP organoid SILK

[0132] ChP organoids were labelled with 50% mol (100% TTR)13Ce-leucine for one week and subsequently cultured in unlabeled media for 24 hours (Fig. 1a). Following labelling, organoids were washed with unlabeled media for 24 hours. CSF-like organoid fluid (iCSF) was collected at multiple time points (1-, 6- and 24 hours). For iCSF collection media was removed, organoids washed with fresh media and iCSF extracted using a pulled glass microcapillary as previously described in Leinonen V, Koivisto AM, Savolainen S, et al. Specifically, iCSF was collected using a pulled glass microcapillary attached to filter and tubing using controlled suction. iCSF was centrifuged at 10,000g for 10min to pellet debris before supernatant was collected, snap frozen in liquid nitrogen and stored at -80°C. iCSF samples were prepared using the same protocol as described for human CSF.ChP Stable Isotope Labelling Kinetics (SILK) in human subjectsNPH cohort

[0133] Individuals with suspected idiopathic NPH (iNPH) were recruited from the specialist hydrocephalus service at the National Hospital for Neurology and Neurosurgery, Queen Square, London. Informed written consent was given. Individuals were prospectively assessed by a board-certified neurologist, and specific symptoms and examination features were recorded according to a pre-specified questionnaire. A collateral history was obtained from their study partner and where possible and a timed walk was carried out.

[0134] Individuals then underwent SILK as previously described in Bateman RJ, Munsell LY, Morris JC, Swarm R, Yarasheski KE, Holtzman DM. Quantifying CNS protein production and clearance rates in humans using in vivo stable isotope labelling, immunoprecipitation, and tandem mass spectrometry. Nature medicine. 2006;12(7):856.Control cohort

[0135] Controls were patients with non-traumatic subarachnoid hemorrhage at the Centre Hospital Montpellier, who had recovered from their acute illness and CSF flow rate had plateaued to within normal clinical parameters.Mass spectrometry analysis of ChP peptides

[0136] T argeted I P-MS of ChP proteins with adjustments for leucine enrichment was carried out. CSF samples (350 pL) were thawed on ice and spiked with 150 ng of yeast enolase internal standard (E6126, Sigma-Aldrich). Proteins were precipitated using 3-volumes of ice-cold acetone at -20°C for 16 hours. After centrifugation at 16,000 g (20 minutes, 4°C), the pellet was air-dried and resolubilised in digest buffer (6 M urea, 2 M thiourea, 2% ASB-14, 200 mM Tris-HCI, pH 8 ). Peptides were reduced with 1 ,4-dithioerythritol and alkylated with iodoacetamide (both Sigma-Aldrich, UK) and digested with 2 pg of Trypsin-LysC (MS grade, Promega) for 16 hours at 37°C . Tryptic peptides were purified by C18 solid-phase extraction (Bond Elute, Agilent Technologies, UK), lyophilised using a centrifugal evaporator and reconstituted in 50 pL 3% acetonitrile (ACN) 0.1% formic acid (FA) for UPLC-MS / MS analysis.

[0137] Targeted analysis of the ChP protein SILK panel was performed on an Acquity I-Class PLUS UPLC system coupled to a Xevo TQ-XS mass spectrometer operated in positive electrospray ionisation (ESI+) mode (Waters, UK). A multiplexed assay was developed and optimised to monitor peptides that were proteotypic for human transthyretin, cystatin-c, apolipoprotein E, albumin and serotransferrin using peptide standards (GenScript, UK). Peptide sequences and their respective ion transitions monitored for unlabelled and labelled ChP proteins are in Supplementary eTable 1.

[0138] Samples were injected (0.5 pL) onto an Acquity Premier peptide ethylene bridged hybrid (BEH) C18 column (300 A, 1.7 pm, 2.1 x 50 mm) held at 50°C and peptides were separated over a 16-minute reverse-phase Acquired data was imported into Skyline (MacCoss Lab software) for peak picking and integration. Peak areas were exported as a .csv file ready for further statistical analysisStatistics

[0139] All statistical analyses were performed using Excel, Prism v.9.5.1 (GraphPad Software), IBM 334 SPSS Statistics (version 27, IBM) or Python software.

[0140] To assess data normality, Shapiro- Wilk tests were performed. Spearman’s correlation analyses were used to assess the association between CSF production rate measured in clinic by LiquoGuard7 and FSR of transthyretin by SILK in vivo. Unpaired t-test was performed to assess if there were significant differences in transthyretin half-life between NPH patients and healthy controls, p < 0.05 was considered statistically significant.ResultsChoroid plexus organoid characteristics

[0141] Human choroid plexus (ChP) organoids form fluid-filled sacks that contain clear colourless fluid by day 42 (Fig. 1a). We have previously shown that human in vitro ChP organoid fluid (‘iCSF’) has a protein profile similar to human CSF - see Pellegrini L, Bonfio C, Chadwick J, Begum F, Skehel M, Lancaster MA. Human CNS barrier forming organoids with cerebrospinal fluid production. Science. 2020;369(6500):eaaz5626. Staining of ChP and CSF transporter markers show that organoids display characteristics of ChP cells by day 40 in vitro (Fig. 1b). Single-cell RNA sequencing between day 27 and day 53 show the enrichment of the ChP epithelial and stromal cells in ChP organoids compared to control, unguided cerebral organoids (Fig. 1e-f). We also show expression of albumin, cystatin-C (Cys-C) and transthyretin (TTHY), with Cys-C and TTHY being highly abundant and albumin minimal (Fig.1g). We selected proteins that were highly abundant in both iCSF and human control CSF, known to be produced by or transported across ChP epithelial cells. These included albumin, apolipoprotein E (ApoE), Cys-C, serotransferrin and TTHY. Since some of these proteins are also constituents of organoid maturation media, we used differences in sequence homology and targeted proteomics to differentiate human from bovine proteins (Fig. 1d). Albumin peptides in iCSF consisted mostly of bovine albumin, indicating that albumin was transported from the media across the media / iCSF barrier. TTHY in iCSF was almost exclusively human, indicating that it was translated by organoid cells.Human subject characteristics

[0142] Fifteen individuals were recruited to measure protein kinetics using SILK; ten individuals with suspected NPH, four individuals recovering from subarachnoid hemorrhage (SAH-controls) and one individual with late-onset Alzheimer’s Disease (AD). Subject characteristics are summarized in Table 1.

[0143] NPH participants were older than controls (NPH: 75, (71-78) vs 57 (46-65) years old; p = 0.0012) with different sex distributions. All NPH patients had consistent motor signs and symptoms and mild cognitive impairment (MMSE: median 28; IQR 26-28; n=9) (Table 1).None had CSF biomarker support for amyloidosis. All individuals had imaging features of disproportionately enlarged subarachnoid-space hydrocephalus. Five / 10 NPH patients underwent pre- and post-shunt computed tomography (CT) imaging, showing a mean ventricular volume reduction of 27.5 ml (-13.11%) (Fig. 6). Seven suspected NPH patients improved in gait and / or cognition after CSF diversion. Two did not respond; one participant died of unrelated causes before outcome determination.Table 1 | Demographic and biomarker data for NPH and control groups (pSAH)Controls (post Alzheimer’s diseaseNPH(n = 10)SAH)(n = 4) (n=1)Age at LP (years)a 75 (71 -78) 57 (46-65) 67No. Male (%) 80 (8 / 10) 0 0No. Caucasian (%) 90 (9 / 10) NA NA MMSE (points)a 28 (26-28)(n = 9)b NA NA Baseline 10 m walk11 (10-23)(n = 9)b NA NAtime (seconds)Number responding7 NA NAto shuntingNo. Executive50 (5 / 10) NA NA dysfunction (%)No. Episodic memory70 (7 / 10) NA NA problems (%)No. Language20 (2 / 10) NA NA impairment (%)No. Gait disturbance100 (10 / 10) NA NA(%)No. Parkinsonism (%) 20 (2 / 10) NA NANo. Positive for falls44 (n = 9)b NA NA(%)No. Urinary80 (8 / 10) NA NA incontinence (%)No. Cerebellar signs11 (n = 9)b NA NA(%)No. Eye movement33 (n = 9)b NA NA abnormalities (%)No. Supranuclear13 (n = 8)b NA NAgaze palsy (%)No. Pyramidal signs11 (n = 9)b NA NA(%)CSF production rate84 (62-89) NA NA (ml / hr)aClinical CSF A 42 / 4O 0.118 (0.082-(ratio)a 0.129)(n = 9)ba: median and interquartile ranges are shown, b: where data was missing, the number of subjects for which the data was available is indicated in parenthesis, LP: lumbar puncture, MMSE: mini-mental state examination, NPH: Normal pressure hydrocephalus, NA: not available.ChP protein kinetics in human choroid plexus organoids

[0144] Using SILK, the tracer-to-tracee ratio (TTR) of peptides corresponding to the ChP-related proteins of interest allowed capture of precise turnover in ChP lysate (reflecting intracellular turnover) and organoid fluid iCSF (reflecting extracellular turnover). Plasma-derived albumin and serotransferrin (data not shown).

[0145] are captured, alongside ChP-derived TTHY, Cys-C and ApoE (data not shown).

[0146] The highest Fractional Synthesis Rates (FSRs) in iCSF and lysate are observed in Cys-C and TTHY followed by ApoE (Table 2). This is around 20-50 fold higher than the plasma derived proteins albumin and serotransferrin (data not shown). This indicated these proteins were rapidly translated and secreted by the ChP, suggesting they make a plausible contribution to CSF synthesis.

[0147] By contrast, plasma-derived proteins show minimal labeling in organoid fluid during the 24 hour pulse phase, with albumin TTR at -1.5% (FSR 0.004 % / hr) and serotransferrin TTR <0.4% (FSR -0.01 % / hr), indicating low synthesis. In organoid lysate, albumin TTR was -10% (FSR 0.8 % / hr), suggesting some synthesis by ChP organoid cells, while serotransferrin TTR was 0.15% (FSR 0.002 % / hr), indicating negligible synthesis. As expected, the FCR of all proteins is extremely low (<0.1 % / hr) in organoid lysate and iCSF, indicating that no significant protein clearance occurs. This information provided proof of principle in the ChP SILK method, and informed the timing of sample collection in vivo, suggesting that early frequent CSF draws would be needed to capture ChP protein turnover.ChP protein kinetics in vivo

[0148] Within the 72 hours studied, we captured the FSR and FCR rates of TTHY, Cys-Cand ApoE in NPH / controls. In both groups TTHY had highest FSR, followed by Cys-C and ApoE (Table 2); control FSRs were similar to rates observed in vitro. Serotransferrin and albumin had much lower FSR, continuing to rise at 72 hours meaning only synthesis rate could be measured. Controls exhibited significantly higher FSR than NPH for TTHY (both ALG and TSE peptides) and serotransferrin (p<0.05) with a trend for higher FSR in controls for all proteins studied.

[0149] FCR was captured for TTHY, Cys-C and ApoE. For TTHY, FCR was ~10-fold lower in NPH than controls (ALG peptide: p<0.01; TSE peptide: p<0.01). For comparison, the kinetic curves of all five proteins within single NPH and control subjects are shown in Fig. 3a-b.Comparing ChP kinetics in ventricular and lumbar CSF

[0150] Since control CSF was ventricular and not lumbar, we investigated whether the site of CSF collection could confound SILK measurements. The FSR and FCR values for TTHY are compared between subarachnoid haemorrhage (SAH) control subjects (yellow-ventricular) and a control with late-onset AD (LOAD) who donated lumbar CSF (dark green-lumbar), showing strong alignment of their kinetic curves despite their CSF compartmental differences (Fig. 3c). This makes it unlikely that differences found in controls versus NPH could be due to differences in kinetics between CSF compartments.Relating protein kinetics to measured CSF flow

[0151] In NPH subjects, transthyretin FSR for both peptides monitored was found to positively and significantly correlate with CSF production measured in-clinic by LiquoGuard7 (p=0.037, p 189 =0.738 for ALG peptide and p=0.042, p=0.683 for TSE peptide) (Fig.3d). No association was observed between CSF production rate measured by LiquoGuard7 and FSR of Cys-C, serotransferrin, albumin or ApoE (p values = 0.5518, 0.8737, 0.8024 and 0.4194, 192 respectively). This suggests that the FSR of TTHY in CSF could be a biomarker reflective of CSF production.Discussion

[0152] We introduce a novel SILK method to measure synthesis and clearance of ChP proteins in human CSF both in vivo and in vitro, revealing significant impairment of CSF clearance in NPH compared to controls. We also identify a previously unreported disruption of ChP synthetic function, suggesting a feedback mechanism that may regulate CSF production in humans.

[0153] Given the lack of definitive pathological hallmark of NPH15 and ongoing debate about its classification as a distinct disease entity (see Espay AJ, Da Prat GA, Dwivedi AK, et al. Deconstructing normal pressure hydrocephalus: ventriculomegaly as early sign ofneurodegeneration. Annals of neurology. 2017;82(4):503-513), we aimed to determine whether differences in protein kinetics could provide objective biomarkers in this population. Using a targeted panel of ChP proteins we tracked: 1) synthesis of proteins from ChP epithelial cells secreted into CSF; 2) transport of peripherally-derived proteins across the blood-CSF barrier, and 3) the clearance / reabsorption of proteins from CSF.

[0154] Human ChP organoids were used to determine which proteins were most abundantly expressed by ChP epithelial cells. Based on previous work using untargeted mass spectrometry of organoid CSF and human CSF14, paired with single-cell RNA sequencing to confirm which cells proteins derived from, we confirmed that TTHY, Cys-C and ApoE were abundantly expressed and rapidly translated by ChP epithelium. Importantly, we found similar results in control human CSF. In contrast, we found extremely low turnover rates in vitro and in vivo for albumin and serotransferrin, suggesting they are likely produced peripherally by the liver and then transported across the blood-CSF barrier.

[0155] The SILK method provides a robust in vivo approach to quantitate choroid plexus (ChP) synthetic function and its capacity to transport proteins across the blood-CSF barrier. These findings were recapitulated in ChP organoids, with broadly similar synthesis (FSR) rates, underscoring the utility of this in vitro model for studying ChP physiology, diseases of abnormal CSF dynamics (e.g., ChP tumors, idiopathic intracranial hypertension) and therapeutic drug delivery into the CNS.

[0156] Using SILK, we also developed a method to quantitate CSF protein clearance or resorption. Of the proteins identified, TTHY was of particular interest as it’s abundantly produced by the ChP17 (see Sousa JC, Cardoso I, Marques F, Saraiva MJ, Palha JA. Transthyretin and Alzheimer's disease: where in the brain? Neurobiology of aging.2007;28(5):713-718.), is rapidly translated and does not readily cross the blood-CSF barrier. Physiologically, TTHY is involved in transporting the thyroid hormone thyroxine into the brain along with retinol binding hormone (see Liz MA, Coelho T, Bellotti V, Fernandez-Arias Ml, Mallaina P, Obici L. A narrative review of the role of transthyretin in health and disease. Neurology and therapy. 2020;9(2):395). The two primary sites of TTHY synthesis are the liver (see Felding P, Fex G. Cellular origin of prealbumin in the rat. Biochimica et Biophysica Acta (BBA)-General Subjects. 1982;716(3):446-449) and the ChP20 which give rise to TTHY in plasma and CSF, respectively. However, there is an 11-fold difference in the mRNA levels and a 13-fold difference in the speed of synthesis between the two regions, with faster production occurring in the ChP21. We next considered whether TTHY clearance would be a good marker of CSF bulk flow clearance or whether it had other routes for removal. Unlike amyloid beta (A ), which may be cleared via the coordinated action of several, removal of TTHY does not appear to be aided by transport proteins. A limited number of studies assessing the ability of TTHY to cross the blood-brain barrier (BBB) suggested that TTHY cancross the BBB but this likely only occurs in the brain-to-blood direction.

[0157] The most striking difference between NPH and control subjects is the clearance rate of TTHY, which is -10 times lower in NPH. Although it did not reach significance, a similar trend was seen for Cys-C. This indicates that CSF protein turnover is ~10 fold longer in NPH, suggesting impaired CSF protein turnover, possible fluid and protein stasis and greater potential for protein aggregation and / or post-translational modification.

[0158] Our findings are consistent with the literature suggesting a mechanism of failed CSF clearance in NPH. Imaging injected intrathecal contrast has suggested a clearance deficit but is not widely used and has ethical limitations. The conventional view of brain fluid clearance and waste removal supports clearance via three routes; via arachnoid granulations into the dural venous sinuses; via nasal lymphatics into the cervical lymph nodes (CLNs) and via transporters / receptors located in the ChP epithelium. Recent studies have characterised meningeal lymphatic vessels. Removal of cellular waste products in the CNS is partly fulfilled by the CSF-ISF exchange paravascular (glymphatic) route. This route is comprised of a network of perivascular channels which drain into the meningeal lymphatic vasculature or the dural sinuses. Evidence from MRI studies support that glymphatic clearance of brain fluid is impaired in individuals with NPH. Combined dysfunction of the glymphatic and meningeal systems may promote ventricular reflux. We speculate that impaired glymphatic and meningeal function may result in reduced ChP protein / TTHY clearance.

[0159] We also observed significant differences in ChP protein secretion into CSF in NPH compared to controls. FSR of TTHY and serotransferrin were significantly lower in NPH, providing in vivo evidence of reduced epithelial cell protein translation (TTHY) and reduced active transport across the ChP (serotransferrin). This may indicate that a feedback mechanism exists to regulate ChP function in humans.

[0160] Finally, we identified a significant relationship between TTHY FSR and measured volume of CSF production. TTHY was chosen as a marker due to its well-established role as a ChP derived protein with rapid synthesis, its specificity to the ChP, and its limited ability to cross the blood-CSF barrier. These properties make TTHY an ideal candidate for reflecting ChP synthetic activity and CSF dynamics. By correlating TTHY FSR with CSF production, we provide a novel, physiologically relevant surrogate marker for tracking CSF flow in vivo. This finding further highlights the potential of this SILK method fortracking CSF dynamics in NPH and other conditions characterised by disrupted CSF homeostasis.

[0161] ChP-protein SILK could be a useful clinical diagnostic marker of NPH. Prolonged diagnostic lumbar drainage is currently the gold standard diagnostic test to identify individuals likely to clinically respond to CSF diversion. However, this method requires -three day admission to hospital, making it costly / burdensome. The integration of stable isotope labelling prior to large volume CSF tap offers a less invasive, more objective approach to diagnosingclearance failure, in addition to clinical evidence of gait improvement. This could provide a more accurate predictive test of clinical responders who might benefit from shunting, at lower cost. The ChP SILK method could also be used to interrogate alterations to CSF flow in other diseases such as idiopathic intracranial hypertension, and ventriculomegaly occurring in response to intrathecal disease modifying therapies. These promising applications warrant further investigation.

[0162] In conclusion, our SILK method provides a valuable novel tool for quantifying ChP function and CSF protein dynamics in vivo and in vitro. ChP organoids recapitulate human CSF protein synthesis rates, highlighting the utility of this model in studying diseases of CSF disruption and evaluating intrathecal drug delivery. The identification of TTHY as a potential kinetic biomarker of CSF flow in vivo offers a novel approach for quantitating CSF flow in the diagnosis and monitoring of NPH and other disorder of CSF flow. Additionally, there is a significant potential for a kinetic protein biomarker that reflects CSF flow to advance other areas of neuroscience — notably in intrathecal therapeutics, where variations in CSF flow dynamics are likely to critically influence drug delivery, clinical efficacy and drug safety.

Claims

CLAIMS1. A method for measuring the synthesis rate (SR) of a biomolecule in a test subject, comprising:(a) provision of a cerebrospinal fluid (CSF) sample obtained from the test subject, wherein the sample was taken at a first time period following exposure to a labeled amino acid under conditions which allowed incorporation of the labeled amino acid into the biomolecule;(b) detecting the amount of a biomolecule labeled with the labeled amino acid in the sample; and(c) either:a. calculating the synthesis rate (SR) from the ratio of labeled biomolecule in the CSF sample obtained from the test subject to a control sample, wherein the control sample is obtained from a healthy subject following exposure to the labeled amino acid under identical conditions to the test subject; orb. repeating step (a) at second time point and calculating the synthesis rate (SR) from the ratio of labeled biomolecule to unlabeled biomolecule determined by step (a) in the biological sample of a test subject over time.

2. A method for measuring the clearance rate (CR) of a biomolecule in a test subject, comprising:(a) provision of a cerebrospinal fluid sample obtained from the test subject, wherein the sample was taken following exposure to a labeled amino acid under conditions which allowed incorporation of the labeled amino acid into the biomolecule during synthesis for a first time period followed by exposure to an unlabeled amino acid under conditions which allowed incorporation of the unlabeled amino acid into the biomolecule during synthesis for a second time period;(b) detecting the amount of the biomolecule labeled with the labelled amino acid and the amount of unlabeled biomolecule in a biological sample obtained from the test subject;(c) provision of one or more samples from the test subject at a later time period during which the test subject is only exposed to unlabeled amino acid and detecting the amount of biomolecule labeled with the labeled amino acid and the amount of the29biomolecule not labeled with the amino acid in a biological sample obtained from the test subject; and(d) calculating the clearance rate (CR) from the ratio of labeled biomolecule to unlabeled biomolecule determined by step (b) and (c).

3. The method according to claim 1 or 2, wherein the biomolecule is a choroid plexus (ChP) protein, preferably selected from the group consisting of transthyretin (TThyrR), apolipoprotein E (APOE), cystatin C (Cys-C), serotransferrin, albumin, Serpin F1, FSTL 1, IGFBP2, Clusterin, GSTP1, ENPP2, TUBA1A, UCHL1, PEBP1, CFL1, PARK7, SOD1, or variants thereof.

4. The method according to any one of claims 1 to 3, wherein the label is a stable nonradioactive isotope selected from 13C or 15N, optionally wherein the labeled amino acid is 13C Leucine, 13C Arginine, or 13C Lysine.

5. The method for measuring synthesis rate according to any one of claims 1, 3 or 4, wherein the sample(s) are taken up to 28 hours following exposure to a labeled amino acid.

6. The method for measuring clearance rate, wherein the first sample is taken from 0 to 72 hours following exposure to a labeled amino acid and one or more later samples are taken thereafter.

7. The method according to any one of claims 1 to 6, further comprising(a) comparing the SR and / or CR of a biomolecule in a test subject to a reference SR and / or CR representing a known disease or health status, and / or(b) comparing the SR and / or CR of a biomolecule in a test subject between two different time periods.

8. A method of determining abnormal cerebrospinal fluid flow in a test subject and / or an increased risk of a developing a disorder associated with abnormal cerebrospinal fluid flow, the method comprising:(a) determining the synthesis rate and / or clearance rate of a biomolecule in the test subject in accordance with the method of any one of claims 1 to 7;(b) comparing the synthesis rate and / or clearance rate to a reference value or to the synthesis and / or clearance rate of a control healthy subject, wherein the process for30obtaining the synthesis and / or clearance rate of the control healthy subject is identical to that of the test subject; and(c) determining abnormal cerebrospinal fluid flow and / or increased risk of a developing a disorder associated with abnormal cerebrospinal fluid flow in the test subject if the clearance rate and / or synthesis rate is lower than the reference value or at least 2-fold lower than the control healthy subject.

9. A method of diagnosing a disorder associated with abnormal cerebrospinal fluid flow in a subject suspected of having a disorder associated with abnormal cerebrospinal fluid flow comprising:(a) determining the synthesis rate and / or clearance rate of a biomolecule in the subject in accordance with the method of any one of claims 1 to 7;(b) comparing the synthesis rate and / or clearance rate to a reference value or to the synthesis and / or clearance rate of a control healthy subject, wherein the process for obtaining the synthesis and / or clearance rate of the control healthy subject is identical to that of the subject suspected of having a disorder associated with abnormal cerebrospinal fluid flow; and(c) diagnosing a disorder associated with abnormal cerebrospinal fluid flow in the subject if the clearance rate and / or synthesis rate is lower than the reference value or at least 2-fold lower than the control healthy subject.

10. Use of the method according to any one of claims 1 to 7 for diagnosing abnormal CSF flow in a test subject.

11. Use of the method according to any one of claims to 1 to 7 in determining whether the subject has an increased risk of developing or diagnosing a disorder associated with abnormal CSF flow, optionally wherein the disorder is selected from the group consisting of: hydrocephalus, idiopathic intracranial hypertension, Subarachnoid haemorrhage, Vestibular Schwanoma, CSF hypotension and Impaired CSF flow following intrathecal therapeutics.

12. Use according to claim 11, wherein the disorder is hydrocephalus, optionally wherein the disorder is normal pressure hydrocephalus.

13. A method of treating a disorder associated with abnormal cerebrospinal fluid flow in a subject identified as having abnormal cerebrospinal fluid flow in accordance with claim 8 or diagnosed as having a disorder associated with abnormal cerebrospinal fluid flowin accordance with claim 9, wherein the method comprises performing lumbar or ventriculoperitoneal shunting.

14. A method for measuring the synthesis rate (SR) of a biomolecule in vitro, comprising:(a) provision of an in vitro choroid plexus organoid fluid sample, wherein the sample was taken at a first time period following exposure to a labeled amino acid under conditions which allowed incorporation of the labeled amino acid into the biomolecule; (b) detecting the amount of a biomolecule labeled with the labeled amino acid in the sample; and(c) repeating steps (a) and (b) at second time point and calculating the synthesis rate (SR) from the ratio of labeled biomolecule to unlabeled biomolecule determined by step over time.

15. A method for measuring the clearance rate (CR) of a biomolecule in vitro, comprising:(a) provision of an in vitro choroid plexus organoid fluid sample, wherein the sample was taken following exposure to a labeled amino acid under conditions which allowed incorporation of the labeled amino acid into the biomolecule during synthesis; for a first time period followed by exposure to an unlabeled amino acid under conditions which allowed incorporation of the unlabeled amino acid into the biomolecule during synthesis for a second time period;(b) detecting the amount of a biomolecule labeled with the amino acid and a biomolecule not labeled with the labeled amino acid in a biological sample obtained from the test subject;(c) provision of one or more samples at a later time period during which the test subject is only exposed to unlabeled amino acid and detecting the amount of the biomolecule labeled with the amino acid and the biomolecule not labeled with the labelled amino acid in a biological sample obtained from the test subject; and(d) calculating the clearance rate (CR) from the ratio of labeled biomolecule to unlabeled biomolecule determined by step (b) and (c).

16. Use of a one or more proteins selected from the group consisting of transthyretin (TThyrR), apolipoprotein E (APOE), cystatin C (Cys-C), serotransferrin, albumin, Serpin F1, FSTL 1, IGFBP2, Clusterin, GSTP1, ENPP2, TUBA1A, UCHL1, PEBP1, CFL1 , PARK7, SOD1 , or variants thereof as a biomarker for: determining cerebrospinal fluid flow, identifying as subject at risk of developing a disorderassociated with abnormal cerebrospinal fluid flow, or diagnosing a disorder associated with cerebrospinal fluid flow.

17. Use according to claim 16, wherein the disorder is selected from the group consisting of: hydrocephalus, idiopathic intracranial hypertension, Subarachnoid haemorrhage, Vestibular Schwanoma, CSF hypotension and Impaired CSF flow following intrathecal therapeutics.33

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