WO2023028196A1

WO2023028196A1 - Poly(gp) dipeptide repeat protein assays and methods of treatment employing such assays

Info

Publication number: WO2023028196A1
Application number: PCT/US2022/041466
Authority: WO
Inventors: Ramakrishna Bharadwaj BOYANAPALLI; Adrian Michael Isaacs
Original assignee: Wave Life Sciences Ltd.; UCL Business Ltd.
Priority date: 2021-08-26
Filing date: 2022-08-25
Publication date: 2023-03-02

Abstract

The present disclosure relates to highly sensitive assays for detecting poly(GP) dipeptide repeat proteins as well as methods of using such assays in the development and administration of therapeutic interventions for diseases associated with C9orf72 poly(GP) dipeptide repeat expansion.

Description

POLY(GP) DIPEPTIDE REPEAT PROTEIN ASSAYS AND METHODS OF TREATMENT EMPLOYING SUCH ASSAYS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.: 63/237,303, filed August 26, 2021, the content of which is incorporated by reference in its entirety, and to which priority is claimed.

FIELD OF THE INVENTION

BACKGROUND

A GGGGCC repeat expansion in the first intron of C9orf72 is the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) accounting for 38% and 25% of familial cases respectively. Healthy individuals most commonly have two repeats, while people with a C9orf72 repeat expansion (C9FTD/ALS) can carry hundreds to thousands of repeats. The repeats are transcribed in both sense and antisense direction, leading to the formation of RNA aggregates termed RNA foci. In addition, repeat-associated non-ATG (RAN) translation of the repeat expansion leads to the production of dipeptide repeat proteins (DPRs) from both sense and antisense transcripts producing five different dipeptide species, poly(GA), poly(GP), poly(GR), poly(PR) and poly (PA). Antisense Oligonucleotides (ASOs) targeting the repeat expansion or C9orf72 transcripts have been shown to reduce both RNA foci and DPR levels in human iPSC- neurons and C9orf72 mouse models.

In order to progress therapies from the bench to the bedside, biomarkers of disease that can reflect target engagement are needed. An important breakthrough was the discovery that poly(GP) can be detected in the cerebrospinal fluid (CSF) of people with C9FTD/ALS using Meso Scale Discovery (MSD) immunoassays, indicating its potential as a target engagement biomarker. While levels of poly(GP) in CSF have not been found to correlate with clinical disease markers or neurofilament light (NfL) and heavy (NfH) chain protein levels in CSF, a non-disease specific biomarkers of neurodegeneration, ASO treatment of mice models has been shown to lead to durable, decreased poly(GP) levels both in brain tissues and mouse CSF, indicating that CSF poly(GP) levels could be used as a pharmacodynamic biomarker if a sufficiently sensitive and specific assay could be developed.

SUMMARY

In certain embodiments, the present disclosure provides a method for determining a measure of the concentration of poly(GP) dipeptide repeat proteins in a cerebral spinal fluid (CSF) sample containing poly(GP) dipeptide repeat proteins, comprising: (a) contacting the CSF sample containing poly(GP) dipeptide repeat proteins with a plurality of capture probes, the capture probes being linked to one or more capture ligands that specifically bind to the poly(GP) dipeptide repeat proteins, and incubating to allow binding of the capture ligands to the poly(GP) dipeptide repeat proteins; (b) contacting the product of (a) with a plurality of detection probes that specifically bind to the poly(GP) dipeptide repeat proteins, and incubating to allow binding of the detection probes to the poly(GP) dipeptide repeat proteins, the detection probes each being linked to a detectable moiety; (c) washing the product of (b) to remove unbound detection probe; (d) detecting the detectable moieties remaining after the wash of step (c); and (e) comparing the signal associated with the detectable moieties to a concentration reference standard, thereby determining a measure of the concentration of poly(GP) dipeptide repeat proteins in the sample.

In certain embodiments, the capture ligand is an antibody. In certain embodiments, the capture ligand is a monoclonal antibody. In certain embodiments, the capture ligand is the monoclonal antibody TALS 828.179.

In certain embodiments, the detection probe is an antibody. In certain embodiments, the detection probe is monoclonal antibody. In certain embodiments, the detection probe is polyclonal antibody. In certain embodiments, the detection probe is a polyclonal antibody raised against a GP(32) antigen. In certain embodiments, the detection probe is the polyclonal antibody GP57, the polyclonal antibody GP60, or a combination of the polyclonal antibodies GP57 and GP60. In certain embodiments, the detection probe is a polyclonal antibody raised against a GP(8) antigen. In certain embodiments, the detection probe is the polyclonal antibody GP6834.

In certain embodiments, the method exhibits a lower limit of quantitation (LLOQ) of about Ipg/ml to about 200 pg/ml. In certain embodiments, the present disclosure provides method of treatment comprising: (a) determining a measure of the concentration of poly(GP) dipeptide repeat proteins in a cerebral spinal fluid (CSF) sample containing poly(GP) dipeptide repeat proteins obtained from said subject prior to and subsequent to administration of a therapeutic directed to engagement of C9orf72 repeat containing transcripts, the determination comprising: (i) contacting the CSF sample containing poly(GP) dipeptide repeat proteins with a plurality of capture probes, the capture probes being linked to one or more capture ligands that specifically bind to the poly(GP) dipeptide repeat proteins, and incubating to allow binding of the capture ligands to the poly(GP) dipeptide repeat proteins; (ii) contacting the product of (i) with a plurality of detection probes that specifically bind to the poly(GP) dipeptide repeat proteins, and incubating to allow binding of the detection probes to the poly(GP) dipeptide repeat proteins, the detection probes each being linked to a detectable moiety; (iii) washing the product of (ii) to remove unbound detection probe; (iv) detecting the detectable moieties remaining after the wash of step (iii); and (v) comparing the signal associated with the detectable moieties to a concentration reference standard, thereby determining a measure of the concentration of poly(GP) dipeptide repeat proteins in the CSF sample; and (b) continuing to administer the therapeutic if the concentration of poly(GP) dipeptide repeat protein in the CSF sample is reduced after administration of the therapeutic relative to the concentration prior to administration of the therapeutic.

In certain embodiments, the measure of the concentration exhibits an LLOQ of about Ipg/ml to about 200 pg/ml. BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1C depict assessment of curve fitting. Figure 1A depicts assessment of heteroscedasticity of data carried out by plotting standard deviation of assay signals (AEB) from the calibrator curve standards from 7 independent assays, against the calibrator concentration (pg/ml). Figure IB depicts that to calculate weighting, Linear regression was applied after plotting Log(standard deviation of assay signals) plotted against Log(mean of assay signals) and the slope of the line (k) used in the formula: Weighting= 1/Y2k. Figure 1C shoes that curves were recalculated using 4PL and 5PL, with no weighting, 1.9474, or 2 weighting. Curve fits were assessed using criteria that cumulative Relative errors% (RE%) and CV% for calibrators +/- 15%, and RE% and CV% for anchor points (1 pg/ml) +/- 20%. When 4PL 1/Y2 was used for curve fitting, all calibrator points passed these criteria and 4PL 1/Y2 was therefore chosen.

Figure 2 depicts comparison of monoclonal and polyclonal anti-poly(GP) antibodies in Simoa homebrew assays. Homebrew Simoa assay conditions were improved using different capture antibodies and detector antibodies (*). mGP = monoclonal poly(GP) antibody (TALS 828.179). GP57*-60* is a combination of two custom polyclonal antibodies ‘GP57’ and ‘GP60’. GP6834 is an alternative custom made poly(GP) antibody. Dashed lines show predicted LLOQs for each assay respectively (mGP +mGP*, mGP +GP57*-60*, mGP + GP6834*), calculated using Quanterix assay developer tool, after running 6-point standard curves using GST-GP32 as standard.

Figures 3A-3B depict transfer of poly(GP) assay onto Simoa HD-X. Figure 3 A shows that the effect of sample diluents was assessed by comparing signal/noise (S/N) using control human CSF spiked with 25 pg/ml GST-GP32 standard, diluted 1 in 2 with different Quanterix diluents. Samples were run in duplicate on a single 2 step Simoa assay (HD-X), using mGP + GP57*-60* Homebrew assay. Figure 3B depicts a standard curve produced from the improved mGP + GP57*-60* HD-X Simoa assay, using GST-GP32 as standard. LLOQ at 1.17 pg/ml shown by dashed line.

Figures 4A-4J depict CSF poly(GP) Simoa assay qualification. 10-point standard curves ranging from 200 to 1 pg/ml and 3 quality control (QC) samples (15 pg/ml, 75 pg/ml 140pg/ml) were prepared using GST-GP32 peptide and measured on 7 independent assays. Figure 4A depicts the coefficient variation (CV) measured for each standard, calculating first the CV for 3 initial assays (green dot) and then comparing subsequent assays to the average signal from those 3 assays. Red dotted line at +/- 20% acceptance level. Figure 4B depicts the difference from total (DFT) calculated for each standard across 7 independent assays. DFT = % difference between predicted concentration and actual concentration of standards. Red dotted lines at +/- 20% acceptance level. Figure 4C depicts CVs for QC samples across 7 independent assays. Green dot displaying the CV from the 3 initial assays. Red dotted lines at +/- 20% acceptance level. Figure 4D depicts the Simoa assay signal, average enzyme on bead (AEB) measured for QCs prepared by 2 different analysts, each prepared 3 independent sets of QCs and ran on one assay each. Figure 4E depicts DFTs calculated for QC samples ran on 7 independent assays. Red dotted lines at +/- 20% acceptance level. Figure 4F depicts intra-plate variability assessed by measuring QCs in 3 different positions across a single assay plate. Figure 4G depicts human C9orf72 CSF donor sample (QC4) measured on 4 independent assays showing high precision. Furthermore, QC4 underwent 0, 1, 2, or 3 freeze-thaw cycles prior to measure in a single assay. Red dotted lines at +/- 20% acceptance level from the fresh measured QC4 sample. Figure 4H depicts dilutional parallelism measured using 6 C9orf72 CSF samples serially diluted, using 1 in 2 dilution as anchor. Predicted concentration % error was calculated comparing the adjusted predicted concentration at each dilution to the concentration of the 1 in 2 diluted sample (set to 100%). Red dotted lines denote +/- 30% from the expected predicted concentration. Figure 41 is a photo of CSF spiked with hemolysate ranging from 1% to 0.000064%. Figure 4J depicts CSF spiked with hemolysate and serial diluted to give range of equivalent % hemolysate. CSF was also spiked with 50pg/ml GST-GP32 and poly(GP) concentration measured using Simoa assay. Three sets were assayed and % error in predicted concentration was plotted for each sample. Red dotted lines at +/- 20% from expected poly(GP) concentration.

Figures 5A-5B depict dilutional parallelism. CSF from six C9orf72 expansion positive donors was measured either neat, 1:2, 1:4, 1:8 and 1:16 diluted in diluent A. The mean AEB from duplicate measures was used to predict concentration at each dilution. In Figure 5A the neat sample concentration was used as anchor and the % error was calculated comparing the adjusted predicted concentration at each dilution to the concentration of the neat sample. In Figure 5B the 1 :2 diluted sample used as anchor instead. Red dotted lines denote +/- 30% from the expected predicted concentration.

Figures 6A-6C depict hemoglobin interference. Control CSF was spiked with hemolysate and serial diluted to give range of equivalent % hemolysate. CSF was also spiked with either 5 pg/ml (Figure 6A) or 50 pg/ml GST-GP32 (Figure 6B) and poly(GP) concentration measured using Simoa assay. Three sets at each GST-GP32 concentration were assayed and % error in predicated concentration was plotted for each sample. Red dotted lines at +/- 20% from expected poly(GP) concentration. Figure 6C shows visual appearance of CSF after hemolysate spiking.

Figures 7A-7D depict poly(GP) levels in CSF from C9orf72 expansion carriers. Poly(GP) levels in CSF from 25 presymptomatic C9orf72 expansion carriers, 15 symptomatic C9orf72 carriers and 15 healthy aged matched controls were measured using our improved Simoa HD-X assay. In Figure 7A Signal/Noise (S/N) was calculated by dividing the average AEB signal from duplicate measures of 40 C9orf72 expansion carriers, by the average AEB signal of CSF from all 15 healthy controls (plotted here as 1). C9orf72 expansion carriers had poly(GP) assay signals distinct from healthy controls, with all Signal/Noise values above 8. Figure 7B depicts comparison of poly(GP) levels in presymptomatic and symptomatic C9orf72 expansion carriers. Each data point is the average from a duplicate measure from each donor, with bar at mean for each group. Lower Limit of Quantification (LLOQ) at 1 pg/ml is shown with dotted line. There is no statistical difference in poly(GP) levels between presymptomatic and symptomatic C9orf72 expansion carriers (Mann-Whitney U test). Figure 7C depicts that age of onset plotted against poly(GP) pg/ml in CSF for 15 symptomatic C9orf72 expansion carriers, ns = not significant, no correlation found (Spearman r). Figure 7D depicts age at donation plotted against CSF poly(GP) levels. Red dot indicates high poly(GP) CSF case, which if removed, data no longer correlates significantly with age at donation (new p=0.0522).

Figures 8A-8B depict analysis of poly(GP) CSF levels with clinical features. Figure 8A depicts no difference between female and male C9orf72 expansion carriers in CSF poly(GP) levels. Figure 8B depicts age at visit for symptomatic C9orf72 expansion carriers plotted against CSF poly(GP) levels.

Figure 9 depicts analysis of brain volume with poly(GP) CSF levels. Left-hand side; total brain volume, temporal lobe, parietal lobe, occipital lobe and frontal lobe volumes against poly(GP) CSF levels from C9orf72 expansion carriers (N=38). Right-hand side analysis of same regions from symptomatic C9orf72 expansion carriers (N=14).

Figures 10A-10D depict analysis of plasma biomarkers from matched CSF donors. Plasma samples from 5 controls, 10 presymptomatic and 8 symptomatic C9orf72 expansion carriers whom also had poly(GP) CSF measured. Figure 10A shows that plasma NfL levels were significantly higher in symptomatic carriers compared to presymtomatic carriers (Kruskal Wallis and Dunn’s multiple comparisons, ** p<0.01). Figure 20B shows that no correlation was observed between plasma NfL levels and CSF poly(GP) levels in the available matched samples from 8 symptomatic cases. Figure 10C depicts raw AEB signals from Simoa assay adapted to measure poly(GP) in plasma. No difference was observed between controls or C9orf72 expansion carriers. Figure 10D depicts raw AEB signals from plasma samples plotted against matched samples CSF poly(GP) levels.

DETAILED DESCRIPTION

Definitions

As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001.

As used herein in the present disclosure, unless otherwise clear from context, (i) the term “a” or “an” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; (iii) the terms “comprising”, “comprise”, “including” (whether used with “not limited to” or not), and “include” (whether used with “not limited to” or not) may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; (iv) the term “another” may be understood to mean at least an additional/second one or more; (v) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (vi) where ranges are provided, endpoints are included.

The term “nucleic acid”, as used herein, includes any nucleotides and polymers thereof. The term “polynucleotide”, as used herein, refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) or a combination thereof. These terms refer to the primary structure of the molecules and, thus, include double- and single-stranded DNA, and double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA comprising modified nucleotides and/or modified polynucleotides, such as, though not limited to, methylated, protected and/or capped nucleotides or polynucleotides. The terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and/or modified nucleobases; nucleic acids derived from sugars and/or modified sugars; and nucleic acids derived from phosphate bridges and/or modified intemucleotidic linkages. The term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified intemucleotidic linkages. Examples include, and are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. Unless otherwise specified, the prefix poly- refers to a nucleic acid containing 2 to about 10,000 nucleotide monomer units and wherein the prefix oligo- refers to a nucleic acid containing 2 to about 200 nucleotide monomer units.

The term "oligonucleotide" refers to a polymer or oligomer of nucleotides, and may contain any combination of natural and non-natural nucleobases, sugars, and intemucleotidic linkages.

Oligonucleotides can be single-stranded or double-stranded. A single-stranded oligonucleotide can have double-stranded regions (formed by two portions of the singlestranded oligonucleotide) and a double-stranded oligonucleotide, which comprises two oligonucleotide chains, can have single-stranded regions for example, at regions where the two oligonucleotide chains are not complementary to each other. Example oligonucleotides include, but are not limited to structural genes, genes including control and termination regions, self-replicating systems such as viral or plasmid DNA, single-stranded and doublestranded RNAi agents and other RNA interference reagents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics, supermirs, aptamers, antimirs, antagomirs, U1 adaptors, triplex-forming oligonucleotides, G- quadruplex oligonucleotides, RNA activators, immuno-stimulatory oligonucleotides, and decoy oligonucleotides.

Oligonucleotides of the present disclosure can be of various lengths. In particular embodiments, oligonucleotides can range from about 2 to about 200 nucleosides in length. In various related embodiments, oligonucleotides, single-stranded, doublestranded, or triple-stranded, can range in length from about 4 to about 10 nucleosides, from about 10 to about 50 nucleosides, from about 20 to about 50 nucleosides, from about 15 to about 30 nucleosides, from about 20 to about 30 nucleosides in length. In certain embodiments, the oligonucleotide is from about 9 to about 39 nucleosides in length. In certain embodiments, the oligonucleotide is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides in length. In certain embodiments, the oligonucleotide

at least 4 nucleosides in length. In certain embodiments, the oligonucleotide

at least 5 nucleosides in length. In certain embodiments, the oligonucleotide is at least 6 nucleosides in length. In certain embodiments, the oligonucleotide is at least 7 nucleosides in length. In certain embodiments, the oligonucleotide is at least 8 nucleosides in length. In certain embodiments, the oligonucleotide is at least 9 nucleosides in length. In certain embodiments, the oligonucleotide is at least 10 nucleosides in length. In certain embodiments, the oligonucleotide is at least 11 nucleosides in length. In certain embodiments, the oligonucleotide is at least 12 nucleosides in length. In certain embodiments, the oligonucleotide is at least 15 nucleosides in length. In certain embodiments, the oligonucleotide is at least 15 nucleosides in length. In certain embodiments, the oligonucleotide is at least 16 nucleosides in length. In certain embodiments, the oligonucleotide is at least 17 nucleosides in length. In certain embodiments, the oligonucleotide is at least 18 nucleosides in length. In certain embodiments, the oligonucleotide is at least 19 nucleosides in length. In certain embodiments, the oligonucleotide is at least 20 nucleosides in length. In certain embodiments, the oligonucleotide is at least 25 nucleosides in length. In certain embodiments, the oligonucleotide is at least 30 nucleosides in length. In certain embodiments, each nucleoside counted in an oligonucleotide length independently comprises a nucleobase comprising a ring having at least one nitrogen ring atom. In certain embodiments, each nucleoside counted in an oligonucleotide length independently comprises A, T, C, G, or U, or optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G or U.

As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In certain embodiments, an active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In certain embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

The term “pharmaceutically acceptable salt”, as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In certain embodiments, pharmaceutically acceptable salt include, but are not limited to, nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. In certain embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fiimarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. In certain embodiments, a provided compound comprises one or more acidic groups, e.g., an oligonucleotide, and a pharmaceutically acceptable salt is an alkali, alkaline earth metal, or ammonium (e.g., an ammonium salt of N(R)3, wherein each R is independently defined and described in the present disclosure) salt. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In certain embodiments, a pharmaceutically acceptable salt is a sodium salt. In certain embodiments, a pharmaceutically acceptable salt is a potassium salt. In certain embodiments, a pharmaceutically acceptable salt is a calcium salt. In certain embodiments, pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate. In certain embodiments, a provided compound comprises more than one acid groups, for example, an oligonucleotide may comprise two or more acidic groups (e.g., in natural phosphate linkages and/or modified intemucleotidic linkages). In certain embodiments, a pharmaceutically acceptable salt, or generally a salt, of such a compound comprises two or more cations, which can be the same or different. In certain embodiments, in a pharmaceutically acceptable salt (or generally, a salt), all ionizable hydrogen (e.g., in an aqueous solution with a pKa no more than about 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2; in certain embodiments, no more than about 7; in certain embodiments, no more than about 6; in certain embodiments, no more than about 5; in certain embodiments, no more than about 4; in certain embodiments, no more than about 3) in the acidic groups are replaced with cations. In certain embodiments, each phosphorothioate and phosphate group independently exists in its salt form (e.g., if sodium salt, -O-P(O)(SNa)-O- and -O-P(O)(ONa)-O-, respectively). In certain embodiments, each phosphorothioate and phosphate intemucleotidic linkage independently exists in its salt form (e.g., if sodium salt, -O-P(O)(SNa)-O- and -O-P(O)(ONa)-O-, respectively). In certain embodiments, a pharmaceutically acceptable salt is a sodium salt of an oligonucleotide. In certain embodiments, a pharmaceutically acceptable salt is a sodium salt of an oligonucleotide, wherein each acidic phosphate and modified phosphate group (e.g., phosphorothioate, phosphate, etc.), if any, exists as a salt form (all sodium salt).

As used herein, the term “subject” or “test subject” refers to any organism to which a or to which a compound (e.g., an oligonucleotide) or composition is administered in accordance with the present disclosure e.g., for experimental, diagnostic, prophylactic and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In certain embodiments, a subject is a human. In certain embodiments, a subject may be suffering from and/or susceptible to a disease, disorder and/or condition.

As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. A base sequence which is substantially identical or complementary to a second sequence is not fully identical or complementary to the second sequence but is mostly or nearly identical or complementary to the second sequence. In certain embodiments, an oligonucleotide with a substantially complementary sequence to another oligonucleotide or nucleic acid forms duplex with the oligonucleotide or nucleic acid in a similar fashion as an oligonucleotide with a fully complementary sequence. In addition, one of ordinary skill in the biological and/or chemical arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and/or chemical phenomena.

An individual who is “susceptible to” a disease, disorder and/or condition is one who has a higher risk of developing the disease, disorder and/or condition than does a member of the general public. In certain embodiments, an individual who is susceptible to a disease, disorder and/or condition is predisposed to have that disease, disorder and/or condition. In certain embodiments, an individual who is susceptible to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder and/or condition. In certain embodiments, an individual who is susceptible to a disease, disorder and/or condition may exhibit symptoms of the disease, disorder and/or condition. In certain embodiments, an individual who is susceptible to a disease, disorder and/or condition may not exhibit symptoms of the disease, disorder and/or condition. In certain embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In certain embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

As used herein, the term “therapeutic agent” in general refers to any agent that elicits a desired effect (e.g., a desired biological, clinical, or pharmacological effect) when administered to a subject. In certain embodiments, an agent, e.g., a dsRNAi agent, is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In certain embodiments, an appropriate population is a population of subjects suffering from and/or susceptible to a disease, disorder or condition. In certain embodiments, an appropriate population is a population of model organisms. In certain embodiments, an appropriate population may be defined by one or more criterion such as age group, gender, genetic background, preexisting clinical conditions, prior exposure to therapy. In certain embodiments, a therapeutic agent is a substance that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms or features of a disease, disorder, and/or condition in a subject when administered to the subject in an effective amount. In certain embodiments, a “therapeutic agent” is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In certain embodiments, a “therapeutic agent” is an agent for which a medical prescription is required for administration to humans. In certain embodiments, a therapeutic agent is a provided compound, e.g., a provided oligonucleotide.

As used herein, the term “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen. In certain embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition. In certain embodiments, a therapeutically effective amount is administered in a single dose; in certain embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely prevent one or more symptoms or features of a disease, disorder, and/or condition (i.e., “preventative treatment” or “prophylactic treatment”) or to alleviate, ameliorate, relieve, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition (i.e. “therapeutic treatment”). In either case, treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition. In certain embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.

By “target analyte” is meant the composition, e.g., apoly(GP) dipeptide repeat protein, to be either detected, measured, quantified, or evaluated in the context of the assay. A target analyte can be contained in a sample. In certain embodiments, the sample will be a liquid sample, e.g., a CSF sample.

The term “capture probe,” as used herein, means a moiety to which a target analyte can be conjugated, captured, attached, bound, or affixed. In some embodiments, a target analyte is conjugated, captured, attached, bound, or affixed to a capture probe by a capture ligand. Suitable capture probes include, but are not limited to, beads (e.g., magnetic beads (e.g., paramagnetic beads), silica beads, or hydrogel beads), nanotubes, polymers, or the like. In some embodiments, a droplet holds zero or one capture probes. In other embodiments, a droplet may hold more than one capture probe.

The term “capture ligand,” as used herein, means a moiety that is capable of specifically binding to or otherwise specifically associating with a capture probe or a target analyte. A capture ligand may be conjugated, captured, attached, bound, or affixed to a capture probe. For example, in some embodiments, a capture ligand is an antibody (e.g., a fidl-length antibody (e.g., an IgG, IgA, IgD, IgE, or IgM antibody) or an antigen-binding antibody fragment (e.g., an scFv, an Fv, a dAb, a Fab, an Fab', an Fab'2, an F(ab')2, an Fd, an Fv, or an Feb)), an aptamer, an antibody mimetic (e.g., an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, an avimer, a DARPin, a fynomer, a Kunitz domain peptide, a monobody, or a nanoCLAMP), an antibody IgG binding protein (e.g., protein A, protein G, protein L, or recombinant protein A/G), a polypeptide, a nucleic acid, or a small molecule.

The terms “bead,” “particle,” and “microsphere,” as used interchangeably herein, mean a small discrete particle. Suitable beads include, but are not limited to, magnetic beads (e.g., paramagnetic beads), plastic beads, ceramic beads, glass beads, silica beads, polystyrene beads, methylstyrene beads, acrylic polymer beads, carbon graphited beads, titanium dioxide beads, latex or cross-linked dextrans such as SEPHAROSE beads, cellulose beads, nylon beads, cross-linked micelles, and TEFLON® beads. In some embodiments, spherical beads are used, but non-spherical or irregularly-shaped beads may be used.

The term “detection probe,” as used herein, means any molecule, particle, or the like that is capable of specifically binding to or otherwise specifically associating with a target analyte or another molecule that binds to or otherwise associates with the target analyte (e.g., another detection probe). For example, in some embodiments, a detection probe is an antibody (e.g., a full-length antibody (e.g., an IgG, IgA, IgD, IgE, or IgM antibody) or an antigen-binding antibody fragment (e.g., an scFv, an Fv, a dAb, a Fab, an Fab', an Fab'2, an F(ab')2, an Fd, an Fv, or an Feb)), an aptamer, an antibody mimetic (e.g., an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, an avimer, a DARPin, a fynomer, a Kunitz domain peptide, a monobody, or a nanoCLAMP), a molecularly-imprinted polymer, a receptor, a polypeptide, a nucleic acid, or a small molecule.

The term “detectable moiety,” as used herein, means a moiety that can produce a detectable signal. For example, in some embodiments, a detectable moiety is or comprises an enzymatic label (e.g., beta-galactosidase, horseradish peroxidase, glucose oxidase, and alkaline phosphatase), a fluorescent label, a radioactive label, or a metal label. In particular embodiments, the detectable moiety is beta-galactosidase.

The term “non-covalent affinity binding pair” refers to a pair of moieties that bind and form a non-covalent complex. Exemplary non-covalent affinity binding pairs include, without limitation, biotin-biotin binding protein (e.g., biotin-streptavidin and biotin-avidin), ligand-receptor, antigen-antibody or antigen binding fragment, hapten-anti- hapten, and immunoglobulin (Ig) binding protein-Ig. The members of a non-covalent affinity binding pair will have a binding affinity suitable for the assay sensitivity desired. For example, the members of an affinity binding pair can bind with an equilibrium dissociation constant (KD or Kd) of about 10^-5 M, 10^-6 M, 10^-7 M, 10^-8 M, 10^-9 M, IO^-10 M, 10^-11 M, 10“¹² M, 10“¹³ M, 10“¹⁴ M, 10“¹⁵ M, or lower.

A first moiety “specifically binds” (or grammatical variants thereof) a second moiety if the first moiety (e.g., a detection probe) binds to the second moiety (e.g., a target analyte or an immobilized target analyte) with specificity sufficient to differentiate between the second moiety and other components or contaminants of the test sample. The binding is generally sufficient to remain bound under the conditions of the assay, including wash steps to remove non-specific binding, although in some embodiments, wash steps are not desired; i.e., for detecting low affinity binding partners. In some embodiments, a first moiety specifically binds to a second moiety with an equilibrium dissociation constant (KD or Kd) of about 10“⁵ M, 10“⁶ M, 10“⁷ M, 10“⁸ M, 10“⁹ M, 10-10 M, 10^-11 M, 10“¹² M, 10“¹³ M, 10“¹⁴ M, 10^-15 M, or lower.

Assay Methods

In certain embodiments, the present disclosure is directed to a sensitive, qualified poly(GP) assay using single molecule array (Simoa) technology. As outlined in detail herein, the Simoa platform measures immuno-complexes bound to microscopic beads that are isolated in arrays of microwells, where the wells are large enough for a single bead. Using digital detection and Poisson distribution for quantification, the Simoa platform enables single molecule detection. Exemplary Simoa platforms and strategies useful in the context of the present disclosure are presented in U.S. Patent No. 8,236,574, which is incorporated by reference herein in its entirety.

In certain embodiments, the present disclosure is directed to assay methods, e.g., Simoa assay methods, for determining a measure of the concentration of poly(GP) dipeptide repeat proteins in a sample, e.g., a cerebral spinal fluid (CSF) sample, containing poly(GP) dipeptide repeat proteins.

In certain embodiments, the methods of the present disclosure comprise contacting the sample, e.g., CSF sample, containing poly(GP) dipeptide repeat proteins with a plurality of capture probes. For example, but not by way of limitation, the capture probes can directly bind poly(GP) dipeptide repeat proteins or the capture probes can comprise one or more capture ligands, where the capture ligand specifically binds to the poly(GP) dipeptide repeat proteins. In certain embodiments the capture ligand is directly or indirectly linked to the capture probe. In certain embodiments, the assay methods of the present disclosure can comprise incubating the sample, e.g., CSF sample, with the capture probe to allow binding of the capture probe, e.g., via a linked capture ligand, to the poly(GP) dipeptide repeat protein.

In certain embodiments, the methods of the present disclosure comprise contacting the capture probes, subsequent to incubation with a sample, e.g., a CSF sample, with a plurality of detection probes. In certain embodiments the detection probes are linked, directly or indirectly, to a detectable moiety. In certain embodiments, the detection probes are capable of specifically binding to poly(GP) dipeptide repeat proteins that have been bound by the capture probes, e.g., via an interaction with a capture ligand. In certain embodiments, the assay methods of the present disclosure can comprise incubating the detection probes with the capture probes that have been exposed to a sample, e.g., a CSF sample, to allow binding of the detection probes to the poly(GP) dipeptide repeat proteins bound to the capture probes.

In certain embodiments, the assay methods of the present disclosure can comprise a wash step after the detection probes are contacted to the capture probes, where the capture probes have previously been contacted with a sample, e.g., a CSF sample. In certain embodiments, the wash step is sufficient to substantially remove detection probe that has not bound the poly(GP) dipeptide repeat proteins bound to the capture probes.

In certain embodiments, the assay methods of the present disclosure comprise a detection step. For example, but not by way of limitation, the detection step can comprise detecting the detectable moieties linked to the detection probes. In certain embodiments the detection step will detect the detectable moieties remaining after the wash of step that substantially removes any detection probe that has not bound the poly(GP) dipeptide repeat proteins bound to the capture probes.

In certain embodiments, the assay methods of the present disclosure comprise comparing the signal associated with the detectable moieties to a concentration reference standard. In certain embodiments, such a comparison allows for the determination of a measure of the concentration of poly(GP) dipeptide repeat proteins in the sample.

In certain embodiments of the assay methods of the present disclosure, the capture ligand is an antibody. In certain embodiments, the capture ligand is a monoclonal antibody. In certain embodiments, the capture ligand is the monoclonal antibody TALS 828.179.

In certain embodiments of the assay methods of the present disclosure, the detection probe is an antibody. In certain embodiments, the detection probe is monoclonal antibody. In certain embodiments, the detection probe is polyclonal antibody. In certain embodiments, the detection probe is a polyclonal antibody raised against a GP(32) antigen. In certain embodiments, the detection probe is the polyclonal antibody GP57, the polyclonal antibody GP60, or a combination of the polyclonal antibodies GP57 and GP60. In certain embodiments, the detection probe is a polyclonal antibody raised against a GP(8) antigen In certain embodiments, the detection probe is the polyclonal antibody GP6834.

In certain embodiments, the assay methods of the present disclosure exhibit a lower limit of quantitation (LLOQ) of about Ipg/ml to about 200 pg/ml. In certain embodiments, the assay methods of the present disclosure exhibit an LLOQ of about 1 pg/ml to about 150 pg/ml. In certain embodiments, the assay methods of the present disclosure exhibit an LLOQ of about Ipg/ml to about 100 pg/ml. In certain embodiments, the assay methods of the present disclosure exhibit an LLOQ of about Ipg/ml to about 50 pg/ml. In certain embodiments, the assay methods of the present disclosure exhibit an LLOQ of about Ipg/ml to about 25 pg/ml. In certain embodiments, the assay methods of the present disclosure exhibit an LLOQ of about Ipg/ml to about 10 pg/ml. In certain embodiments, the assay methods of the present disclosure exhibit an LLOQ of about Ipg/ml to about 5 pg/ml. In certain embodiments, the assay methods of the present disclosure exhibit an LLOQ of about Ipg/ml.

Treatment Methods

In certain embodiments, the present disclosure is directed to methods of treating diseases associated with C9orf72 poly(GP) dipeptide repeat expansion. For example, but not by way of limitation, the present disclosure is directed to methods of treatment comprising determining a measure of the concentration of poly(GP) dipeptide repeat proteins in a sample, e.g., a cerebral spinal fluid (CSF) sample, containing poly(GP) dipeptide repeat proteins. In certain embodiments, a therapeutic decision, e.g., to initiate, maintain, increase, decrease, or abstain from administration of a therapeutic directed to engagement of C9orf72 repeat containing transcripts. In certain embodiments, samples, e.g., CSF samples, are obtained from a subject prior to and subsequent to administration of a therapeutic directed to engagement of C9orf72 repeat containing transcripts and a therapeutic decision is made based on a comparison of the concentrations of poly(GP) dipeptide repeat proteins present in the samples. In certain embodiments, the treatment methods of the present disclosure comprise determining the concentration of poly(GP) dipeptide repeat proteins present in a sample, e.g. a sample obtain prior to initiation of treatment with a therapeutic directed to engagement of C9orf72 repeat containing transcripts or a sample obtained after initiation of such treatment, by contacting the sample, e.g., CSF sample, containing poly(GP) dipeptide repeat proteins with a plurality of capture probes. For example, but not by way of limitation, the capture probes can directly bind poly(GP) dipeptide repeat proteins or the capture probes can comprise one or more capture ligands, where the capture ligand specifically binds to the poly(GP) dipeptide repeat proteins. In certain embodiments the capture ligand is directly or indirectly linked to the capture probe. In certain embodiments, the treatment methods of the present disclosure can comprise incubating the sample, e.g., CSF sample, with the capture probe to allow binding of the capture probe, e.g., via a linked capture ligand, to the poly(GP) dipeptide repeat protein.

In certain embodiments, the treatment methods of the present disclosure comprise contacting the capture probes, subsequent to incubation with a sample, e.g., a CSF sample, with a plurality of detection probes. In certain embodiments the detection probes are linked, directly or indirectly, to a detectable moiety. In certain embodiments, the detection probes are capable of specifically binding to poly(GP) dipeptide repeat proteins that have been bound by the capture probes, e.g., via an interaction with a capture ligand. In certain embodiments, the treatment methods of the present disclosure can comprise incubating the detection probes with the capture probes that have been exposed to a sample, e.g., a CSF sample, to allow binding of the detection probes to the poly(GP) dipeptide repeat proteins bound to the capture probes.

In certain embodiments, the treatment methods of the present disclosure can comprise a wash step after the detection probes are contacted to the capture probes, where the capture probes have previously been contacted with a sample, e.g., a CSF sample. In certain embodiments, the wash step is sufficient to substantially remove detection probe that has not bound the poly(GP) dipeptide repeat proteins bound to the capture probes.

In certain embodiments, the treatment methods of the present disclosure comprise a detection step. For example, but not by way of limitation, the detection step can comprise detecting the detectable moieties linked to the detection probes. In certain embodiments the detection step will detect the detectable moieties remaining after the wash of step that substantially removes any detection probe that has not bound the poly(GP) dipeptide repeat proteins bound to the capture probes. In certain embodiments, the treatment methods of the present disclosure comprise comparing the signal associated with the detectable moieties to a concentration reference standard. In certain embodiments, the treatment methods of the present disclosure comprise comparing the signal obtained before and after administration of a therapeutic directed to engagement of C9orf72 repeat containing transcripts. In certain embodiments, such a comparison allows for the determination of a measure of the concentration of poly(GP) dipeptide repeat proteins in the samples or a determination of a relative measure of the concentration of poly(GP) dipeptide repeat proteins in the samples.

In certain embodiments, the treatment method comprises continuing to administer the therapeutic if the concentration of poly(GP) dipeptide repeat protein in the CSF sample is reduced after administration of the therapeutic relative to the concentration prior to administration of the therapeutic. In certain embodiments, the treatment method comprises reducing the dose and/or frequency of administration of the therapeutic if the concentration of poly(GP) dipeptide repeat protein in the CSF sample is reduced after administration of the therapeutic relative to the concentration prior to administration of the therapeutic. In certain embodiments, the treatment method comprises increasing the dosage and/or frequency of administration of the therapeutic if the concentration of poly(GP) dipeptide repeat protein in the CSF sample is maintained or increased after administration of the therapeutic relative to the concentration prior to administration of the therapeutic. In certain embodiments, the treatment method comprises discontinuing administration of the therapeutic if the concentration of poly(GP) dipeptide repeat protein in the CSF sample is maintained or increased after administration of the therapeutic relative to the concentration prior to administration of the therapeutic.

In certain embodiments of the treatment methods of the present disclosure, the capture ligand is an antibody. In certain embodiments, the capture ligand is a monoclonal antibody. In certain embodiments, the capture ligand is the monoclonal antibody TALS 828.179.

In certain embodiments of the treatment methods of the present disclosure, the detection probe is an antibody. In certain embodiments, the detection probe is monoclonal antibody. In certain embodiments, the detection probe is polyclonal antibody. In certain embodiments, the detection probe is a polyclonal antibody raised against a GP(32) antigen. In certain embodiments, the detection probe is the polyclonal antibody GP57, the polyclonal antibody GP60, or a combination of the polyclonal antibodies GP57 and GP60. In certain embodiments, the detection probe is a polyclonal antibody raised against a GP(8) antigen In certain embodiments, the detection probe is the polyclonal antibody GP6834.

In certain embodiments, the treatment methods of the present disclosure exhibit a LLOQ of about Ipg/ml to about 200 pg/ml. In certain embodiments, the treatment methods of the present disclosure exhibit an LLOQ of about Ipg/ml to about 150 pg/ml. In certain embodiments, the treatment methods of the present disclosure exhibit an LLOQ of about Ipg/ml to about 100 pg/ml. In certain embodiments, the treatment methods of the present disclosure exhibit an LLOQ of about Ipg/ml to about 50 pg/ml. In certain embodiments, the treatment methods of the present disclosure exhibit an LLOQ of about Ipg/ml to about 25 pg/ml. In certain embodiments, the treatment methods of the present disclosure exhibit an LLOQ of about Ipg/ml to about 10 pg/ml. In certain embodiments, the as treatment say methods of the present disclosure exhibit an LLOQ of about Ipg/ml to about 5 pg/ml. In certain embodiments, the treatment methods of the present disclosure exhibit an LLOQ of about Ipg/ml.

EXAMPLES

Example 1: Polv(GP) Dipeptide Repeat Assay

This Example describes the development and qualification of a sensitive Simoa assay for poly(GP) DPRs in CSF. The qualified poly(GP) assay was used to analyze CSF from a small cohort of CSF samples provided by GENFI, including 15 healthy controls and 40 C9orf72 expansion carriers. Similar to previously published studies, the present assay was able to distinguish controls and C9orf72 expansion carriers. In this cohort 100% sensitivity and 100% specificity with poly(GP) measured in CSF from all C9orf72 expansion carriers was achieved, while controls either measured below detection (13/15) or below limit of quantification (2/15), determined at 1 pg/ml. C9orf72 expansion carriers had a range of poly(GP) from 3-74 pg/ml, with all positive sample signals at least 8 fold higher than control signals, showing a clear separation of controls from C9orf72 expansion samples. In summary it was shown herein the utility of the instant assay strategy for detecting poly(GP) in the CSF of people with a C9orf72 expansion, with assay reliability sufficient to be used for target engagement analysis, e.g., in clinical trials and therapeutic intervention, where C9orf72 repeat containing transcripts are directly targeted.

Materials and Methods

Participants

Fifty-five participants were recruited from the Genetic FTD Initiative (GENFI), a natural history study of genetic FTD based across 27 sites in Europe and Canada (Rohrer, J. D. et al., Lancet Neurol. 2015 Mar;14(3):253-62). Participants included 15 symptomatic C9orf72 expansion carriers (14 with behavioral variant FTD (bvFTD) and 1 with ALS) (Rascovsky, K. et al., Brain 2011 Sep; 134(9): 2456-2477), presymptomatic C9orf72 expansion carriers and 15 non-carrier relatives, as controls. Pathogenic C9orf72 expansion length was defined as more than 30 repeats. Participants consisted of 23 men and 32 women, with a mean (standard deviation) age of 49.4 (13.9) years old at sample collection. Within the disease groups: presymptomatic C9orf72 expansion carriers, 11 men and 14 women, 41.0 (10) years old and symptomatic C9orf72 expansion carriers, 10 men and 5 women, 64.7 (8.5) years old. 15 healthy controls were recruited over the same time period: 2 men and 13 women, 48.2 (11.2) years old.

All people in the study underwent a clinical assessment consisting of a medical history with the participant and informant, and physical examination, with symptomatic status diagnosed by a clinician who was an expert in the FTD field (Rascovsky, K. et al., Brain 2011 Sep; 134(9): 2456-2477; Gomo-Tempini, M. L. et al., Neurology 2011 Mar 15;76(11): 1006-14; Brooks, B. R., et al., Amyotroph. Lateral Scler. 2000 Dec;l(5):293-9; Armstrong, M. J. et al., Neurology 2013 Jan 29;80(5):496-503.; Hoglinger, G. U. et al., Mov. Disord. 2017 Jun;32(6):853-864). All participants also underwent 3D Tl-weighted MR imaging of the brain. Volumetric measures of whole brain and cortical regions were calculated using a previously described method that uses the geodesic information flow (GIF) algorithm, which is based on atlas propagation and label fusion (Cardoso, M. J. et al., IEEE Trans. Med. Imaging (2015) 2015 Sep;34(9):1976-88).

The study procedures were approved by local ethics committees at each of the participating sites and participants provided informed written consent.

CSF and plasma collection

CSF and plasma were collected, processed and stored in aliquots at -80°C according to standardized procedures.

NfL plasma assay

Plasma NfL concentration was measured in 8 matched symptomatic CSF donors, 10 matched presymptomatic CSF donors and 5 matched healthy control CSF donors using the multiplex Neurology 4-Plex A kit (102153, Quanterix, Lexington, USA) on the Simoa HD-1 Analyzer following manufacturer’s instructions.

Antibodies

Rabbit Polyclonal antibodies, ‘GP57’ and ‘GP60’ were produced in-house using a synthetic polypeptide, GP(32) as antigen. An alternative polyclonal anti-GP antibody ‘GP6834’ was custom made by Eurogentec, using GP(8) as antigen. The monoclonal poly(GP) antibody TALS 828.179 was obtained from the Developmental Studies Hybridoma Bank, deposited by Target ALS Foundation.

Antibody bead conjugation and biotinylation was performed as recommended by Quanterix’s Homebrew Assay Development guide. Briefly, 0.3 mL of Carboxylated Paramagnetic Beads were conjugated with 0.2 mg/mL antibody and 0.3 mg/mL EDC with conjugation performed at 2-8°C. This required 80 pg of input antibody. For each biotinylation 130 pg of antibody was used at 1 mg/mL and a 40: 1 ratio of NHS-PEG4-biotin to antibody.

Assay Improvement

Improvement of the poly(GP) Simoa assay was based on the Quanterix homebrew improvement procedure. The following assay conditions were tested: 2 step assay design vs 3 step assay design, varying detector antibody concentration from 0.3 pg/ml to 1.5 pg/ml, varying SBG concentration from 50 pM to 150 pM, the inclusion of helper beads at different ratios or not at all. Multiple assay combinations were run in parallel to enable selection of improved conditions.

GST-GP32 standard curve was prepared from 2 starting stocks (15000 pg/ml and 1500pg/ml), serially diluting down from both in diluent A to create a 9 point standard curve + blank. High, middle and low QC samples were prepared independently for each assay from a 1500 pg/ml stock of GST-GP32. A positive control human CSF sample from C9orf72 expansion carriers (QC4) was created by pooling a small volume of CSF from the 40 C9orf72 expansion carriers in the GENFI cohort.

Curve fitting

To establish a curve fitting, a workflow previously described was followed (Xiang et al., AAPS J., 2018 Mar 13;20(3):45). Firstly, heteroscedasticity (the unequal variability of a variable across a range of values of a second variable that predicts it) was assessed plotting the standard deviation of the AEB signals from the calibrators from seven assays, against their concentration (Figure 1A). As the data showed heteroscedasticity, weighting was determined by plotting log(SD of signals) against log(mean of signals) (Figure IB). After applying linear regression and determining the slope value (k), weighting was then calculated using the following formula: Weighting = 1/Y^2k = 1.9474. Curves were recalculated using four parameter logistic (4PL) and five parameter logistic (5PL), with no weighting, 1.9474, or 2 weighting. Curve fits were assessed using criteria that relative errors (RE) and CV for calibrators were +/- 15%, and RE and CV for anchor points (1 pg/ml) were +/- 20%. Curve fitting with 4PL 1/Y² was selected as it led to all calibrator points passing these criteria (Figure 1C).

Poly(GP) Simoa assay

The improved Simoa (HD-X) assay using TALS 828.179 monoclonal antibody (mGP) beads as capture and a combination of biotinylated GP57 and GP60 (termed GP57*- 60*) as detector used the following assay conditions: 2 step assay, 0.3 pg/ml detector antibody (GP57*-60*), 50 pM SBG, 150000 assay beads (mGP) with 350000 helper beads.

CSF was thawed on ice and diluted 1 :2 with diluent A (Quanterix). 250 pl per sample was loaded into sample plate to allow for duplicate measures. Analysts were blind to genetic status of samples.

Plasma samples were thawed on ice, prior to centrifiigation at 140000 ref for 15 minutes at room temperature. 125 pl of plasma was then diluted with 125 pl of lysate diluent B (Quanterix) to allow duplicate measure per sample. Standard curve was prepared in lysate diluent B diluted 1 :2 with control human plasma. Analysts were blind to genetic status of samples.

Statistical analysis

Statistical analysis was carried out using GraphPad Prism software. Data was tested for normality prior to appropriate parametric or non-parametric tests. Comparing two groups Mann- Whitney tests were used, for more than two groups Kruskal-Wallis tests and Dunn’s multiple comparisons test were used. To assess correlations between poly(GP) and clinical features Spearman rho and p (two-tailed) values were calculated.

Development of poly(GP) Simoa assay

To develop a sensitive poly(GP) Simoa assay, various assay variables were evaluated using the Simoa HD-1 analyzer. A mouse monoclonal anti-GP antibody (mGP) was tested and a range of affinity purified rabbit polyclonal antibodies (GP57, GP60 and GP6834) raised against different length GP peptides (Table 1). As the long-term goal was to have sufficient antibody quantities for use in a biomarker assay in clinical trials, antibodies GP57 and GP60 were combined, which were both raised against a GP32 peptide. It was found that using the monoclonal antibody as capture and the combined polyclonal antibodies as detector gave the highest signal to noise ratios for the standards and lowest lower limit of quantification (LLOQ) for detection of a GP32 standard peptide (Figure 2). While use of mGP for both capture and detection would have been useful, due to unlimited supply, even after assay improvement the mGP + mGP* assay (where * indicates the biotinylated detector antibody) had over 10-fold lower sensitivity (LLOQ 15.8 pg/ml) than mGP + GP57*-60* (LLOQ 1.04 pg/ml) (Figure 2). As mGP + GP57*-60* showed the highest sensitivity, this assay was taken forward. To ensure compatibility in the long-term, the assay was next transferred to the newer Simoa HD-X platform. It was found that the assay required re-improvement, with the greatest benefit gained from changing the standard curve diluent from lysate diluent B (HD-1) to diluent A (HD-X) (Figure 3A). In addition, streptavidin-P-D-galactosidase (SBG) was lowered from 100 pM to 50 pM for the final HD- X assay, with an LLOQ of 1.17 pg/ml (Figure 3B).

Table 1. Details of polyclonal and monoclonal antibodies tested in Simoa poly(GP) assays. Rabbit polyclonal antibodies were affinity purified prior to biotinylation and testing.

Qualification of Simoa poly(GP) assay For use in clinical trials, this assay was assessed using standard biomarker assay qualification criteria (Table 2).

Table 2. Biomarker assay qualification of poly(GP) Simoa assay.

Precision performance was assessed by analyzing standard curves from 7 independent assays, and was performed independently twice. Coefficient of variation (CV) was <20% for all standard curve points (Figure 4A and Table 3). Difference from total (DFT) (difference between predicted and actual concentration of standards) was below 20% for all standards in 6/7 assays (Figure 4B and Table 4). LLOQ was identified as 1 pg/ml with upper limit of qualification at 200 pg/ml. Quality control (QC) samples were prepared by spiking the standard reference material GST-GP32 into diluent A. Upper QC (150 pg/ml), Middle QC (75 pg/ml) and Lower QC (5 pg/ml) all showed CVs <20% after 7 independent runs (Figure 4C and Table 5). DFTs were below 25% for QCs in 7 assay runs (Figure 4E and Table 6). Intraplate reproducibility was assessed measuring 3 sets of QCs across a plate within a single assay, with CV <5% for all 3 QCs (Figure 4F and Table 7). An endogenous matrix QC sample (QC4) was generated by pooling human CSF from C9orf72 expansion positive donors. Poly(GP) concentration of QC4 was measured in 4 independent assays and the CV was <20% (Figure 4G and Table 8). Reproducibility was farther tested by measurement of QC samples prepared 3 times. This was repeated by a second analyst (Figure 4D and Table 9). CV was <20% for the sets of QCs prepared independently and between the two analysts.

Table 3. Standard curve CV% assessment.

CV% calculated from average AEB values from 3 initial standard curves. Total of 7 assays carried out by 2 independent analysts.

Table 4. Standard curve DFT% assessment.

DFT = difference from total % predicted concentration of standards (pg/ml) versus actual.

Table 5. Assessment of Quality controls (QCs) CV%.

Table 6. Assessment of Quality controls (QCs) DFT%.

Table 7. Intraplate variability assessment of CV%.

Table 8. Reproducibility of matrix control CV%.

Table 9. Reproducibility assessment using independently prepared QCs.

Dilutional parallelism was assessed by running CSF from six C9orf72 expansion positive donors either neat, 1:2, 1:4, 1:8 and 1:16 in diluent A. Poly(GP) was detected above background in all dilutions. Using 1 :2 as an anchor point the average % error of 4 out of 6 samples had <30% error at 1 :4 dilution, passing qualification criteria (Figure 4H). The percentage error increased above 30% for the majority of samples at 1:8 and 1:16 (Table 10 and Figure 5). Samples where run at 1:2 dilution and further assessment was recommended of parallelism within trials with more samples.

Table 10. Dilutional parallelism was assessed by running CSF from six C9orf72 expansion positive donors either neat, 1:2, 1:4, 1:8 and 1:16 in diluent A.

Freeze thaw stability of poly(GP) in CSF was tested using QC4 and measuring poly(GP) after 1, 2, and 3 freeze-thaw cycles. The signal and concentration measured had CVs of 4% and 5% respectively indicating no effect of freeze-thaw on detection of endogenous poly(GP) (Figure 4G and Table 11). The freeze thaw stability of the standard (GST-GP32) was also assessed after 1, 2, or 3 freeze thaw cycles. 8 of the standards passed criteria with CV <20% and DFT <20% (Table 12). The lowest standard point, Ipg/ml gave a higher DFT after 3 freeze thaw cycles, but this is explained by the higher CV in signal measured for the blank in this set of standards, and we therefore concluded that it is unlikely that up to three freeze-thaw cycles affects the signal from GST-GP32.

Table 11. Freeze thaw stability of poly(GP) from human CSF.

Table 12. Freeze-thaw stability of GST-GP32 standard.

During CSF collection it is possible for blood to contaminate the collected CSF. It was tested if hemoglobin interfered with poly(GP) detection. A range of hemolysate concentrations was spiked (Figure 41) into control CSF and spiked with either 5 pg/ml or 50 pg/ml GST-GP32. 5 pg/ml GST-GP32 spiked in CSF was not affected by any of the hemolysate concentrations tested (Figure 6). The measurement of 50 pg/ml GST-GP32 spiked in CSF was inhibited (>20%) by addition of 1% hemolysate (Figure 4J). At this concentration of hemoglobin, the CSF is visibly red (Figure 41), so samples can be excluded from analysis by appearance if required. Note, none of the CSF samples measured in this study had a red or pink appearance.

Measurement of poly(GP) in CSF from C9orf72 expansion carriers using the improved, qualified Simoa assay

This sensitive, qualified assay was used to measure poly(GP) in a cohort of CSF from healthy controls (N=15) and C9orf72 expansion positive donors (N=40). The assay signal from the lowest C9orf72 case had signal/noise 8-fold over the average signal from control samples, showing a clear separation from signals of control CSF (Figure 7A). On average the signal to noise of C9orf72 cases versus controls was 38-fold. Poly(GP) in CSF from healthy donors was below detection level for 13 out of 15 samples or below LLOQ of the assay for the remaining 2 out of 15 cases. As poly(GP) was detected above LLOQ in all cases and in no controls, sensitivity and specificity were both 100%. Poly(GP) measures ranged from 3-74 pg/ml in C9orf72 expansion positive donors. Despite the increased sensitivity of this Simoa assay, just as previously reported the levels of poly(GP) were not statistically different between presymptomatic and symptomatic C9orf72 expansion positive donors (p=0.1348 Mann- Whitney test), although it was observed herein the same trend observed by others towards higher levels in symptomatic cases (Figure 7B). No difference was found in poly(GP) levels between male and female C9orf72 expansion positive donors (Figure 8A). No correlation was found between CSF poly(GP) levels and age of onset of symptomatic C9orf72 expansion positive donors (N=15) (Figure 7C). Interestingly there was a significant, moderate positive correlation (r= 0.3643) between age at donation and poly(GP) measured in CSF, analyzing all 40 C9orf72 expansion positive cases (Figure 7D). However, if the case with the highest poly(GP) level is removed from analysis the P value changes to P=0.0522.

Where data was available correlations between CSF poly(GP) levels and both total brain and lobar volumes were also tested. No correlation was found, analyzing all C9orf72 expansion carriers or selecting symptomatic cases only (Figure 9), consistent with a previous report.

Plasma neurofilament light chain (NfL) is a known biomarker of neurodegeneration. Plasma levels of NfL were measured in 18 of the C9orf72 expansion carrier CSF donors (including 8 symptomatic donors). As expected, plasma NfL levels were significantly higher in symptomatic carriers (Figure 10A). No correlation was found with CSF poly(GP) and plasma NfL levels analyzing the small sample of 8 symptomatic cases (Figure 10B).

The poly(GP) Simoa assay was next applied to analysis of plasma. Despite the high sensitivity of the Simoa platform poly(GP) in plasma were not detected. Signals were below LLOQ and there was no difference between control and C9orf72 positive signals (Figure 10C). The two cases of plasma from C9orf72 expansion carriers which had higher AEB signals, were not the same donors with higher than average CSF poly(GP), and there was no correlation between plasma AEB signal and poly(GP) measured in matched CSF samples (Figure 10D). There is a predicted 200-fold drop in concentration of NfL measured between CSF and plasma. The levels of poly(GP) in CSF were on average 13 pg/ml, so if a similar reduction is observed for poly(GP) an alternative platform capable of detecting in femtogram range maybe required to measure poly(GP) in plasma.

Claims

WHAT IS CLAIMED IS:

1. A method for determining a measure of the concentration of poly(GP) dipeptide repeat proteins in a cerebral spinal fluid (CSF) sample containing poly(GP) dipeptide repeat proteins, comprising:

(a) contacting the CSF sample containing poly(GP) dipeptide repeat proteins with a plurality of capture probes, the capture probes being linked to one or more capture ligands that specifically bind to the poly(GP) dipeptide repeat proteins, and incubating to allow binding of the capture ligands to the poly(GP) dipeptide repeat proteins;

(b) contacting the product of (a) with a plurality of detection probes that specifically bind to the poly(GP) dipeptide repeat proteins, and incubating to allow binding of the detection probes to the poly(GP) dipeptide repeat proteins, the detection probes each being linked to a detectable moiety;

(c) washing the product of (b) to remove unbound detection probe;

(d) detecting the detectable moieties remaining after the wash of step (c); and

(e) comparing the signal associated with the detectable moieties to a concentration reference standard, thereby determining a measure of the concentration of poly(GP) dipeptide repeat proteins in the sample.

2. The method of claim 1, wherein the capture ligand is an antibody.

3. The method of claim 2, wherein the capture ligand is a monoclonal antibody.

4. The method of claim 3, wherein the capture ligand is the monoclonal antibody TALS

828.179.

5. The method of claim 1 , wherein the detection probe is an antibody.

6. The method of claim 5, wherein the detection probe is monoclonal antibody.

7. The method of claim 5, wherein the detection probe is polyclonal antibody.

8. The method of claim 7, wherein the detection probe is a polyclonal antibody raised against a GP(32) antigen.

38 The method of claim 8, wherein the detection probe is the polyclonal antibody GP57, the polyclonal antibody GP60, or a combination of the polyclonal antibodies GP57 and GP60. The method of claim 7, wherein the detection probe is a polyclonal antibody raised against a GP(8) antigen. The method of claim 10, wherein the detection probe is the polyclonal antibody GP6834. The method of claim 1, wherein the method exhibits a lower limit of quantitation (LLOQ) of about Ipg/ml to about 200 pg/ml. A method of treatment comprising:

(a) determining a measure of the concentration of poly(GP) dipeptide repeat proteins in a cerebral spinal fluid (CSF) sample containing poly(GP) dipeptide repeat proteins obtained from said subject prior to and subsequent to administration of a therapeutic directed to engagement of C9orf72 repeat containing transcripts, the determination comprising:

(z) contacting the CSF sample containing poly(GP) dipeptide repeat proteins with a plurality of capture probes, the capture probes being linked to one or more capture ligands that specifically bind to the poly(GP) dipeptide repeat proteins, and incubating to allow binding of the capture ligands to the poly(GP) dipeptide repeat proteins;

(it) contacting the product of (i) with a plurality of detection probes that specifically bind to the poly(GP) dipeptide repeat proteins, and incubating to allow binding of the detection probes to the poly(GP) dipeptide repeat proteins, the detection probes each being linked to a detectable moiety;

(Hi) washing the product of (ii) to remove unbound detection probe;

(zv) detecting the detectable moieties remaining after the wash of step (iii); and

(v) comparing the signal associated with the detectable moieties to a concentration reference standard, thereby determining a measure of the concentration of poly(GP) dipeptide repeat proteins in the CSF sample (b) continuing to administer the therapeutic if the concentration of poly(GP) dipeptide repeat protein in the CSF sample is reduced after administration of the therapeutic relative to the concentration prior to administration of the therapeutic. The method of claim 13, wherein the capture ligand is an antibody. The method of claim 14, wherein the capture ligand is a monoclonal antibody. The method of claim 15, wherein the capture ligand is the monoclonal antibody TALS 828.179. The method of claim 13, wherein the detection probe is an antibody. The method of claim 17, wherein the detection probe is monoclonal antibody. The method of claim 17, wherein the detection probe is polyclonal antibody. The method of claim 19, wherein the detection probe is a polyclonal antibody raised against a GP(32) antigen. The method of claim 19, wherein the detection probe is the polyclonal antibody GP57, the polyclonal antibody GP60, or a combination of the polyclonal antibodies GP57 and GP60. The method of claim 19, wherein the detection probe is a polyclonal antibody raised against a GP(8) antigen. The method of claim 22, wherein the detection probe is the polyclonal antibody GP6834. The method of claim 12, wherein the measure of the concentration exhibits an LLOQ of about Ipg/ml to about 200 pg/ml.