CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application 63/015,991, filed April 27, 2020. The content of the foregoing application is incorporated by reference herein in its entirety.

BACKGROUND

Angelman Syndrome is a genetic disorder caused by deficiency of the maternal UBE3A gene. Loss of UBE3A leads to a clinical presentation consistent with central nervous system dysfunction and is characterized by intellectual disability, developmental delay, speech impairment, seizures and ataxia. An obstacle to the development of effective therapies for treating Angelman syndrome is the lack of methods for monitoring UBE3A levels in human samples.

SUMMARY In one aspect, provided herein are methods of detecting the presence or amount of a UBE3A protein in a sample, the methods include: contacting the sample with an antibody that binds to the UBE3A protein; removing from the sample some or all of the proteins that are not bound to the antibody, resulting in a purified protein preparation; subjecting the purified protein preparation to enzymatic digestion, resulting in a digested peptide preparation; and determining via mass spectrometry the presence or amount of one or more UBE3A peptides in the digested peptide preparation, thereby identifying the presence or amount of the UBE3A protein in the sample.

In some embodiments, the one or more UBE3A peptides include at least one peptide selected from the peptides depicted in Table 17. In some embodiments, the one or more UBE3A peptides include at least one peptide selected from the group consisting of: VFSSAEALVQSFR (SEQ ID NO:3), NLVNDDDAIVAASK (SEQ ID NO:4), VDPLETELGVK (SEQ ID NO:5), and LEMIAMENPADLKK (SEQ ID NO:6). In some embodiments, the methods include detecting one peptide selected from the group consisting of: VFSSAEALVQSFR (SEQ ID NO:3), NLVNDDDAIVAASK (SEQ ID NO:4), VDPLETELGVK (SEQ ID NO:5), and LEMIAMENPADLKK (SEQ ID NO:6). In some embodiments, the methods include detecting two or more peptides selected from the group consisting of: VFSSAEALVQSFR (SEQ ID NO:3), NLVNDDDAIVAASK (SEQ ID NO:4), VDPLETELGVK (SEQ ID NO: 5), and LEMIAMENPADLKK (SEQ ID NO:6). In some embodiments, the methods include detecting peptides VFSSAEALVQSFR (SEQ ID NO:3), NLVNDDDAIVAASK (SEQ ID NO:4), VDPLETELGVK (SEQ ID NO:5), and LEMIAMENPADLKK (SEQ ID NO:6). In some embodiments, the methods further include detecting one or more additional UBE3A peptides in the digested peptide preparation. In some embodiments, enzymatic digestion comprises contacting the purified protein preparation with trypsin. In some embodiments, the antibody that binds to the UBE3A protein competes with the 3E5 antibody for binding to the UBE3A protein. In some embodiments, the antibody that binds to the UBE3A protein comprises the 3E5 antibody. In some embodiments, the antibody that binds to the UBE3A protein is conjugated to beads. In some embodiments, the ratio by volume between the beads and the sample is between about 1:30 to about 1 :80. In some embodiments, the ratio by volume between the beads and the sample is between about 1:40 to about 1:60. In some embodiments, the sample includes about 400 pg/mL or less of the UBE3A protein. In some embodiments, the sample includes about 100 pg/mL or less of the UBE3A protein. In some embodiments, the sample includes about 20 pg/mL or less of the UBE3A protein. In some embodiments, the sample includes about 1 to about 20 pg/mL of the UBE3A protein. In some embodiments, the sample includes about 1 to about 10 pg/mL of the UBE3A protein. In some embodiments, the sample includes about 1 to about 5 pg/mL of the UBE3A protein. In some embodiments, the sample is a human sample. In some embodiments, the sample is a cerebrospinal fluid (CSF) sample. In some embodiments, the sample is a human CSF sample. In some embodiments, the sample is a human CSF sample obtained from a human subject with Angelman syndrome or at risk of developing Angelman syndrome. In some embodiments, identifying the sample as having an undetectable level of the UBE3A protein, a level of the UBE3A protein reduced as compared to healthy subjects, or a level of the UBE3A protein within the range found in subjects with Angelman syndrome identifies a human subject from whom the sample was obtained as having Angelman syndrome or at risk of developing Angelman syndrome.

In another aspect, provided herein are methods of treating a human subject that has Angelman syndrome or is at risk of developing Angelman syndrome, the methods include: identifying the human subject as having Angelman syndrome or being at risk of developing Angelman syndrome according to any of the above embodiments; and administering to the human subject a therapeutic.

In another aspect, provided herein are methods for determining if a therapeutic administered to a human subject that has Angelman syndrome or is at risk of developing Angelman syndrome is an efficacious therapeutic, the methods include: administering one or more doses of the therapeutic to the human subject; and measuring, according to any of the methods of detecting the presence or amount of a UBE3A protein in a sample in the above embodiments, a UBE3A protein level in a biological sample obtained from the human subject after administering the one or more doses of the therapeutic, wherein if the UBE3A protein level in the biological sample is higher than the range of UBE3A protein level found in subjects with Angelman syndrome then the therapeutic is identified as being an efficacious therapeutic.

In another aspect, provided herein are methods for determining if a therapeutic administered to a human subject that has Angelman syndrome or is at risk of developing Angelman syndrome is an efficacious therapeutic, the methods include: measuring, according to any of the methods of detecting the presence or amount of a UBE3A protein in a sample in the above embodiments, a UBE3 A protein level in a first biological sample obtained from the human subject; administering one or more doses of the therapeutic to the human subject; and measuring, according to any of the methods of detecting the presence or amount of a UBE3A protein in a sample in the above embodiments, a UBE3A protein level in a second biological sample obtained from the human subject after administering the one or more doses of the therapeutic, wherein if the UBE3A protein level in the second biological sample is higher than the UBE3A protein level in the first biological sample then the therapeutic is identified as being an efficacious therapeutic.

In another aspect, provided herein are methods of treating a human subject that has Angelman syndrome or is at risk of developing Angelman syndrome, the methods include: administering initial doses of a therapeutic to the human subject, wherein each of the initial doses is in the same amount and is administered at the same dosing interval between doses; measuring, according to any of the methods of detecting the presence or amount of a UBE3A protein in a sample in the above embodiments, a UBE3A protein level in a first biological sample obtained from the human subject after administering the initial doses that is higher than (i) a UBE3A protein level measured in a second biological sample obtained from the human subject prior to administering the initial doses, or (ii) the range of UBE3A protein level found in subjects with Angelman syndrome; and administering further doses of the therapeutic to the human subject, wherein each of the further doses is in the same or lesser amount and at the same or lengthened dosing interval as compared to the initial doses.

In another aspect, provided herein are methods of treating a human subject that has Angelman syndrome or is at risk of developing Angelman syndrome, the methods include: administering initial doses of a therapeutic to the human subject, wherein each of the initial doses is in the same amount and is administered at the same dosing interval between doses; measuring, according to any of the methods of detecting the presence or amount of a UBE3A protein in a sample in the above embodiments, a UBE3A protein level in a first biological sample obtained from the human subject after administering the initial doses that is equal to or higher than a pre-determined threshold UBE3A protein level; and administering further doses of the therapeutic to the human subject, wherein each of the further doses is in the same or lesser amount and at the same or lengthened dosing interval as compared to the initial doses. In another aspect, provided herein are methods of treating a human subject that has Angelman syndrome or is at risk of developing Angelman syndrome, the methods include: administering initial doses of a therapeutic to the human subject, wherein each of the initial doses is in the same amount and is administered at the same dosing interval between doses; measuring, according to any of the methods of detecting the presence or amount of a UBE3A protein in a sample in the above embodiments, a UBE3A protein level in a first biological sample obtained from the human subject after administering the initial doses that is higher than (i) a UBE3A protein level measured in a second biological sample obtained from the human subject prior to administering the initial doses, or (ii) the range of UBE3A protein level found in subjects with Angelman syndrome, but equal to or lower than a pre-determined threshold UBE3A protein level; and administering further doses of the therapeutic to the human subject, wherein each of the further doses is in an increased amount and/or at a shortened dosing interval as compared to the initial doses.

In a further aspect, provided herein are methods of treating a human subject that has Angelman syndrome or is at risk of developing Angelman syndrome, the methods include: administering initial doses of a therapeutic to the human subject, wherein each of the initial doses is in the same amount and is administered at the same dosing interval between doses; measuring, according to any of the methods of detecting the presence or amount of a UBE3A protein in a sample in the above embodiments, a UBE3A protein level in a first biological sample obtained from the human subject after administering the initial doses that is equal to or lower than (i) a UBE3A protein level measured in a second biological sample obtained from the human subject prior to administering the initial doses, or (ii) the range of UBE3A protein level found in subjects with Angelman syndrome; and administering further doses of the therapeutic to the human subject, wherein each of the further doses is in an increased amount and/or at a shortened dosing interval as compared to the initial doses.

In a further aspect, provided herein are methods of treating a human subject that has Angelman syndrome or is at risk of developing Angelman syndrome, the methods include: administering initial doses of a therapeutic to the human subject, wherein each of the initial doses is in the same amount and is administered at the same dosing interval between doses; measuring, according to any of the methods of detecting the presence or amount of a UBE3A protein in a sample in the above embodiments, a UBE3A protein level in a first biological sample obtained from the human subject after administering the initial doses that is equal to or lower than a pre-determined threshold UBE3A protein level; and administering further doses of the therapeutic to the human subject, wherein each of the further doses is in an increased amount and/or at a shortened dosing interval as compared to the initial doses.

In some embodiments of any of the methods of treating a human subject that has Angelman syndrome or is at risk of developing Angelman syndrome or determining if a therapeutic administered to a human subject that has Angelman syndrome or is at risk of developing Angelman syndrome is an efficacious therapeutic, the therapeutic is an antisense oligonucleotide that reduces the level of UBE3A antisense transcript (UBE3A-ATS). In some embodiments, the antisense oligonucleotide is administered to the human subject via intrathecal injection.

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 the invention belongs. The methods and materials described herein are exemplary, and methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description and from the claims. DESCRIPTION OF DRAWINGS

FIG. 1 A is a diagram depicting detection of recombinant UBE3Ausing the Singulex assay. FIG. IB is an enlarged graph of the area marked in FIG. 1A.

FIG. 2 is a diagram showing levels of the UBE3 A protein detected in CSF samples subjected to various detergent conditions. Bars indicate concentration (ng/mL) and dots indicate LOD.

FIG. 3 is a diagram showing levels of the UBE3 A protein detected in CSF samples using the Quanterix assay.

FIG. 4 shows levels of the UBE3A protein detected in CSF samples using Liquid Chromatograph-Mass Spectrometry (LC-MS).

FIG. 5 shows levels of the UBE3A protein detected in aliquots of the same CSF sample using LC-MS.

FIG. 6 shows LC-MS based detection of reduced levels of the UBE3A protein in cells treated with UBE3A shRNA as compared to control. FIGs. 7A and 7B show the correlation between the mass spectrometry signal and the volume of CSF samples analyzed using LC-MS.

DETAILED DESCRIPTION

The present disclosure provides methods of detecting via mass spectrometry the presence or amount of a UBE3A protein in a sample. The methods described herein can be used to detect UBE3A protein in a sample that contains, e.g., less than about 1000 pg/mL (e.g., less than about 900, 800, 700, 600, 500, 400, 300, 100, 50, 20, 15, 10, 5, 1 or 0.5 pg/mL) of the UBE3A protein. The sample can be a human sample (e.g., a human CSF sample, such as a human CSF sample obtained from a human subject having or at risk for developing Angelman syndrome). In some embodiments of the methods described herein, identifying the sample as having an undetectable level of the UBE3 A protein, or a level of the UBE3A protein reduced as compared to healthy subjects identifies a subject from whom that sample was obtained as having or at risk for developing Angelman syndrome. The methods provided herein are also useful for monitoring Angelman syndrome disease progression or the effectiveness of treatments for Angelman syndrome. UBE3A

Ubiquitin Protein Ligase E3A (UBE3A) is an E3 ubiquitin ligase that targets proteins for proteasomal degradation by conjugating ubiquitin to the target proteins. UBE3A is also known as E6AP ubiquitin-protein ligase (E6AP), ANCR, AS, EPVE6AP, HPVE6A, HECT-type ubiquitin transferase E3A, human papillomavirus E6-associated protein, and oncogenic protein-associated protein E6-AP. EGBE3A protein is encoded by the EGBE3A gene which in human resides within the 15ql 1.2-ql3.3 locus that is parentally imprinted in neurons (only the maternal copy is normally active). Mutations in EGBE3A can result in certain neurodevelopmental disorders. For example, Prader-Willi syndrome can result from 15ql l.2-ql3.3 paternal allele deletion, whereas Angelman syndrome can be caused by deletion of the maternal allele. 15ql 1.2-ql3.3 duplication (Dupl5q) syndrome, a genetic cause of autism spectrum disorder, can arise from duplications of the maternal allele.

Mutations or deletions in the EGBE3A gene can result in the absence of the protein in many areas of the brain. The amino acid sequence of the human E1BE3A protein is shown below.

MEKLHQCYWKSGEPQSDDIEASRMKRAAAKHLIERYYHQLTEGCGNEACTNEFCASC P TFLRMDNNAAAIKALELYKINAKLCDPHPSKKGASSAYLENSKGAPNNSCSEIKMNKK GARIDFKDVTYLTEEKVYEILELCREREDYSPLIRVIGRVFSSAEALVQSFRKVKQHT KEELKSLQAKDEDKDEDEKEKAACSAAAMEEDSEASSSRIGDSSQGDNNLQKLGPDDV SVDIDAIRRVYTRLLSNEKIETAFLNALVYLSPNVECDLTYHNVYSRDPNYLNLFIIV

MENRNLHSPEYLEMALPLFCKAMSKLPLAAQGKLIRLWSKYNADQIRRMMETFQQLI T YKVISNEFNSRNLVNDDDAIVAASKCLKMVYYANW GGEVDTNHNEEDDEEPIPESSE LTLQELLGEERRNKKGPRVDPLETELGVKTLDCRKPLIPFEEFINEPLNEVLEMDKDY TFFKVETENKFSFMTCPFILNAVTKNLGLYYDNRIRMYSERRITVLYSLVQGQQLNPY LRLKVRRDHIIDDALVRLEMIAMENPADLKKQLYVEFEGEQGVDEGGVSKEFFQLW E

EIFNPDIGMFTYDESTKLFWFNPSSFETEGQFTLIGIVLGLAIYNNCILDVHFPMW Y RKLMGKKGTFRDLGDSHPVLYQSLKDLLEYEGNVEDDMMITFQISQTDLFGNPMMYDL KENGDKIPITNENRKEFVNLYSDYILNKSVEKQFKAFRRGFHMVTNESPLKYLFRPEE IELLICGSRNLDFQALEETTEYDGGYTRDSVLIREFWEIVHSFTDEQKRLFLQFTTGT DRAPVGGLGKLKMIIAKNGPDTERLPTSHTCFNVLLLPEYSSKEKLKERLLKAITYAK GFGML (SEQ ID NO:1)

Angelman syndrome is a neurodevelopmental disorder characterized by intellectual disability, developmental delay, speech impairment, seizures, ataxia, unusually happy demeanor, and motor deficits, among other symptoms. Angelman syndrome patients commonly carry mutations that render the maternally inherited UBE3A gene non-functional. Human genetic studies revealed that Angelman syndrome is associated with de novo maternal deletions of chromosome 15qll-ql3, paternal chromosome 15 uniparental disomy, or rare imprinting defects that affect the transcription of genes within 15qll-ql3 (Clayton-Smith and Laan, JMed Genet 2003;40:87-95). More recent studies indicate that a failure to inherit a normal maternal copy of the UBE3A gene accounts for 85-90% of all Angelman Syndrome cases. In this regard, specific loss-of-function mutations in the human UBE3A locus have been identified in a subset of affected individuals (Kishino et al., Nature Genetics 15: 70-73, 1997; Matsuura et al., Nature Genetics 15: 74-77, 1997). Phenotype severity is correlated with the type of mutation, with the full deletion of 15ql 1-13 the most severe and point mutations in UBE3A less severe (Gentile et al., J Dev Behav Pediatr. 31(7): 592-601, 2010; Valente et al., Epilepsy Research 105: 234-239, 2013).

Methods of Detection

The present disclosure provides methods of detecting a UBE3A protein in a sample, the methods include obtaining a digested peptide preparation from a purified protein preparation, and subjecting the peptide preparation to mass spectrometry to determine the presence or amount of one or more UBE3A peptides. A purified protein preparation can be obtained by contacting the sample with an antibody that binds to the UBE3A protein and removing from the sample some or all of the proteins that are not bound to the antibody.

Usage of the term “antibody” in this disclosure is meant to cover a whole antibody, a bispecific antibody, a tetravalent antibody, a multispecific antibody, a minibody, a nanobody, and antibody fragments. In some embodiments, the antibody that binds to UBE3A is a whole antibody. UBE3A antibodies including but not limited to those described herein are useful for generating a purified protein preparation. The antibody can be monoclonal or polyclonal. Commercially available polyclonal anti- UBE3A antibodies include, but are not limited to, those from GeneTex (GTX101092), Abeam (abl0488, ab3519, ab235984, abl83869), Proteintech (10344-1-AP), Invitrogen (PA3-843, PA5-12038), and Millipore Sigma (HPA039410, HPA040380,

SAB2102627). Commercially available monoclonal anti-UBE3A antibodies include clone 3E5 (e.g. SAB 1404508 (Millipore Sigma), H00007337-M02 (Abnova)); clone EX-8 (e.g. abl95649 (abeam)); clone ERP7330 (e.g. abl26765 (abeam), ab240033 (abeam), MABC761 (Millipore Sigma)); clone 10H7.1 (e.g. MABS1683 (Millipore Sigma)); clone D10D3 (e.g. #7526 (Cell Signaling Technology)); clone 2F6 (e.g. WH0007337M1 (Millipore Sigma)); and clone 19H14L13 (703785 (Invitrogen)).

The anti-UBE3A antibody 3E5 binds to the following UBE3A sub-sequence: ETF QQLITYKVISNEFN SRNLVNDDDAIVAASKCLKMVYYANVVGGEVDTNHN EEDDEEPIPESSELTLQELLGEERRNKKGPRVDPLETELGVKTLDCR (SEQ ID NO:2).

In some embodiments, the antibody that binds to the UBE3A protein binds to the same epitope as 3E5. In some embodiments, the antibody that binds to the UBE3A protein binds to an epitope within SEQ ID NO:2. In some embodiments, the antibody binds to a different epitope from 3E5. Methods of determining whether a particular antibody binds to the same epitope as a reference antibody are known in the art. A particularly useful method is competitive binding, wherein the ability of the antibody of interest to bind to the UBE3A protein in the presence of the reference antibody is measured. Substantial inability of both antibodies to bind simultaneously indicates that substantially the same epitope is involved. In some embodiments, the antibody competes with the 3E5 antibody for binding to UBE3 A. Methods of producing antibodies that bind to a specific antigen or a specific epitope of an antigen is known in the art. The antibody can be in the form of free antibody in a solution and contacting the sample with the antibody includes mixing the antibody solution with the sample. The antibody can also be conjugated to beads, which are used to contact the sample. Beads suitable for antibody conjugation include, e.g., magnetic beads and agarose-based beads. For example, Protein A/G agarose or magnetic beads, or protein A/G sepharose beads can be used. The antibodies described herein can be bound directly to the beads, or indirectly to a pre-coated ligand on the beads. Methods of conjugating antibodies to beads are known and include, for example, use of dimethyl pimelimidate for crosslinking antibodies to beads. Commercially available kits can also be used, such as those provided by Invitrogen (Dynabeads™ Antibody Coupling Kit). In some embodiments, the beads (e g. magnetic beads) are coated with streptavidin, and a biotinylated UBE3A antibody (e.g., clone 3E5) can be coupled to the beads via streptavidin/biotin interaction.

The sample containing UBE3A protein can be contacted with the UBE3A antibody for an amount of time sufficient to allow for binding between the UBE3 A protein and the antibody. For example, the sample containing UBE3A protein can be incubated (e.g., at or below room temperature) with UBE3A antibody conjugated beads for an amount of time sufficient to allow binding of the UBE3A protein to the antibody conjugated beads. The ratio by volume between the antibody-conjugated beads and the sample containing UBE3A protein can between about 1 :30 to about 1 :80 (e.g. 1:30 to about 1 :70, about 1 :40 to about 1 :60, or about 1:50). Some or all proteins in the sample that are not bound to the antibody can be removed, thereby resulting in a purified UBE3A protein preparation. Removal of unbound proteins can be accomplished by washing the antibody conjugated beads one or more times with a buffer. Any suitable buffer that preferably does not affect the binding between UBE3A protein and the antibody can be used. Digestion

The methods described herein further include subjecting the purified protein preparation to enzymatic digestion, resulting in a digested peptide preparation that contains one or more UBE3A peptides. UBE3A peptides as used herein refers to fragments of the UBE3A protein having a length within the range of, e.g., about 3 to about 100 amino acids. The digestion reaction can be carried out at a suitable temperature (e.g., below, at, or above room temperature) for an amount of time sufficient to generate, for example, one or more UBE3A peptides listed in Table 17. Proteases such as but not limited to trypsin, chymotrypsin, carboxypeptidase, serine proteases, proteinase K, papain, and pepsin can be used for digesting the UBE3A protein. In some embodiments, a trypsin-based digestion solution that contains either free trypsin or immobilized trypsin can be used. In some embodiments, trypsin that is modified to inactivate extraneous chymotryptic activity (e.g. TPCK-treated trypsin) is used.

The digested peptide preparation can contain one or more peptides listed in Table 17. In some embodiments, the digested peptide preparation contains a peptide that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to one of the peptides listed in Table 17. In some embodiments, the digested peptide preparation contains a peptide that is different from one of the peptides listed in Table 17 by one, two or three amino acids. In some embodiments, the digested peptide preparation contains one or more (e.g., 2, 3, or all 4) peptides selected from the group consisting of: VFSSAEALVQSFR (SEQ ID NO:3), NLVNDDDAIVAASK (SEQ ID NO:4), VDPLETELGVK (SEQ ID NO:5), and LEMIAMENPADLKK (SEQ ID NO:6). In some embodiments, the digested peptide preparation contains a peptide that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to one of the peptides selected from the group consisting of SEQ ID NOs 3-6. In some embodiments, the digested peptide preparation contains a peptide that is different by addition or deletion of one, two or three amino acids from one of the peptides selected from the group consisting of SEQ ID NOs 3-6. Mass Spectrometry

The methods described herein further include determining via mass spectrometry the presence or amount of one or more UBE3A peptides in the digested peptide preparation, thereby identifying the presence or amount of the UBE3A protein in the sample. Any suitable mass spectrometry systems described herein or known in the art can be used. For example, Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF), Triple Quadrupole Mass Spectrometry, Quadrupole-Trap Mass Spectrometry, Hybrid Linear Ion Trap Orbitrap Mass Spectrometry, and Quadrupole- Orbitrap Mass Spectrometry. In some embodiments, coupled chromatography - mass spectrometry systems are used, such as Liquid chromatography-mass spectrometry (LC- MS). Liquid chromatography, for example, can separate the multiple UBE3A peptides in the digested peptide preparation, which are then subjected to mass spectrometry to measure the mass-to-charge ratio of charged particles, useful for analyzing e.g. the mass of the peptides, their elemental and isotopic composition. Methods of determining the identity or amount of peptides in a sample based on mass spectrometry signal and intensity are known in the art. Mass analyzers useful in LC-MS systems include, e.g. the quadrupole, time-of-flight (TOF), ion traps, and hybrid quadrupole-TOF (QTOF) analyzers.

Some embodiments of the present disclosure provide detection of the presence or amount of one or more peptides in Table 17 via mass spectrometry. Detection of additional peptides not listed in Table 17, e.g., additional UBE3A peptides are also contemplated herein. In some embodiments, the methods include detection of the presence or amount of at least one peptide (e.g., 2, 3, or all 4 peptides) selected from the group consisting of SEQ ID NOs 3-6.

Sample

Samples that contain UBE3 A protein for use in the methods described herein include any of various types of biological samples that can be isolated and/or derived from a subject (e.g., a human subject). The sample can be isolated and/or derived from any fluid, cell or tissue. In some embodiments, the sample is blood serum, blood plasma, whole blood, lymph, saliva, urine, cerebrospinal fluid (CSF), cell lysates, vitreous fluid, or ocular fluid. Samples containing recombinant UBE3 A proteins are also contemplated herein. In some embodiments, the present disclosure provides methods of detecting the presence or amount of a UBE3A protein in a CSF sample (e.g., a human CSF sample).

A sample of CSF can be obtained from an individual according to known methods. For example, CSF can be collected from an individual through lumbar puncture, with or without accompanying x-ray or CT scans, as well as cisternal puncture and ventricular puncture. CSF can also be collected from a tube that has been placed in the fluid, such as a shunt or a ventricular drain. In some embodiments, the sample is obtained from a subject that is diagnosed as having Angelman syndrome or is suspected of having Angelman syndrome.

The sample of the present disclosure can optionally contain about 1000 pg/mL or less (e.g., about 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 20, 10, 1, 0.5 pg/mL or less) of the UBE3 A protein. In some embodiments, the sample contains about 0.1 to about 1000 pg/mL (e.g., about 0.1 to about 800, about 0.1 to about 600, about 0.1 to about 400, about 0.1 to about 300, about 0.1 to about 200, about 0.1 to about 100, about 0.1 to about 50, about 0.1 to about 20, about 0.1 to about 10, about 0.5 to about 1000, about 0.5 to about 800, about 0.5 to about 600, about 0.5 to about 400, about 0.5 to about 300, about 0.5 to about 200, about 0.5 to about 100, about 0.5 to about 50, about 0.5 to about 20, about 0.5 to about 10, about 10 to about 1000, about 10 to about 800, about 10 to about 600, about 10 to about 400, about 10 to about 300, about 10 to about 200, about 10 to about 100, about 10 to about 50, about 10 to about 20, about 20 to about 1000, about 20 to about 800, about 20 to about 600, about 20 to about 400, about 20 to about 300, about 20 to about 200, about 20 to about 100, about 20 to about 50, about 50 to about 1000, about 50 to about 800, about 50 to about 600, about 50 to about 400, about 50 to about 300, about 50 to about 200, about 50 to about 100, about 100 to about 1000, about 100 to about 800, about 100 to about 600, about 100 to about 400, about 100 to about 300, about 100 to about 200, about 200 to about 1000, about 200 to about 800, about 200 to about 600, about 200 to about 400, about 200 to about 300, about 300 to about 1000, about 300 to about 800, about 300 to about 600, about 300 to about 400, about 400 to about 1000, about 400 to about 800, about 400 to about 600, about 600 to about 1000, about 600 to about 800, about 800 to about 1000 pg/mL) of the UBE3A protein.

The methods of detection described herein are capable of detecting with high sensitivity the presence or amount of low levels of UBE3A protein in a sample. In some embodiments, the methods of detection described herein detect the presence or amount of UBE3A protein in samples that contain 20 pg/mL or less (e.g., 15, 10, 5, 1, 0.5 pg/mL or less) of UBE3A protein.

A sample that is to be subjected to analysis can optionally be spiked with an internal standard, prior to immunoprecipitation and digestion, to normalize for run to run variation and any anomalies in immunoprecipitation and digestion. For example, a known amount of a heavy UBE3A protein (e g., an N15-labeled UBE3A protein) can be added to a sample (e.g., a CSF sample) before the sample is processed (i.e., before immunoprecipitation and digestion).

In addition, a sample that is to be subjected to analysis can optionally be spiked with an internal standard, after immunoprecipitation and digestion but prior to mass spectrometry, to normalize for run to run variation and any anomalies in mass spectrometry. For example, a known amount of one or more heavy synthesized UBE3A peptides (e.g., one or more UBE3A peptides labeled with a C13/N15-C-terminal lysine, such as labeled NLVNDDDAIVAASK (SEQ ID NO:4) and/or labeled VDPLETELGVK (SEQ ID NO: 5)) can be added to a sample preparation after immunoprecipitation and digestion but prior to LC/MS analysis.

In some embodiments, both of the foregoing internal controls (i.e., a first internal standard added prior to immunoprecipitation and digestion combined with a second internal standard added prior to mass spectrometry) are used to maximize reliability of the assay results.

Methods of Diagnosis, Treatment, and Treatment Monitoring

This disclosure provides methods for determining whether a human subject has or is at risk for developing Angelman syndrome. Also provided are methods for monitoring disease progression in a subject diagnosed with Angelman syndrome, or monitoring an Angelman syndrome patient’s response to a treatment. The methods rely on detection of the presence or amount of a UBE3A protein in one or more samples (e.g., any suitable sample described herein) obtained from the subject. In some embodiments, the subject has been identified as having Angelman syndrome or at risk for developing Angelman syndrome based on other diagnostic methods (including but not limited to those described herein). In some embodiments, the subject is undergoing treatment for Angelman syndrome. For example, the UBE3A protein level in the subject undergoing treatment for Angelman syndrome can be monitored by taking one or more samples at two or more different time points during treatment.

The methods include obtaining or having obtained a sample and determining the presence or level of UBE3A in the sample using methods described herein. In some embodiments, the methods include comparing the presence or amount of UBE3A protein in the sample with one or more references, e.g., a control reference that represents a normal level of UBE3A (such as a level in an unaffected subject), or a disease reference that represents a level of the UBE3A protein associated with Angelman syndrome (such as a level in a subject having or at risk for developing Angelman syndrome).

The subject from whom the sample was obtained can be identified as having or at risk of developing Angelman syndrome if the level of UBE3A protein in the sample is undetectable (e.g. using the methods of detection described herein), or if the level of the UBE3A protein is reduced as compared to a control reference that represents a normal level of UBE3A (such as a level in an unaffected subject). The subject can also be identified as having or at risk for developing Angelman syndrome if the level of UBE3A protein in the sample is within the range found in subjects with Angelman syndrome.

Suitable reference values can be determined using methods known in the art.

The reference values can have any relevant form. In some cases, the reference comprises a predetermined value for a meaningful level of UBE3A protein in a sample, e.g., a control reference level that represents a normal level of UBE3A protein, e.g., a level in an unaffected subject or a subject who is not at risk of developing Angelman syndrome, and/or a disease reference that represents a level of UBE3A associated with Angelman syndrome, e.g., a level in a subject having Angelman syndrome.

In some embodiments, the level of UBE3A in a subject is comparable to the level of UBE3A in the disease reference, and the subject has one or more symptoms (e.g., any of the behavioral symptoms or physical appearances described herein) associated with Angelman syndrome, then the subject has Angelman syndrome. In some embodiments, the subject has no overt signs or symptoms of Angelman syndrome, but the level of UBE3A is comparable to the level in the disease reference, then the subject has an increased risk of developing Angelman syndrome.

The predetermined level can be a single cut-off (threshold) value, such as a median or mean, or a level that defines the boundaries of an upper or lower quartile, tertile, or other segment of a clinical trial population that is determined to be statistically different from the other segments. It can be a range of cut-off (or threshold) values, such as a confidence interval. It can be established based upon comparative groups, such as where association with risk of developing Angelman syndrome or presence of Angelman syndrome in one defined group is a fold higher, or lower, (e.g., approximately 2-fold, 4-fold, 8-fold, 16-fold or more) than the risk or presence of Angelman syndrome in another defined group. It can be a range, for example, where a population of subjects (e.g., control subjects) is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group, or into quartiles, the lowest quartile being subjects with the lowest risk and the highest quartile being subjects with the highest risk, or into n-quantiles (i.e., n regularly spaced intervals) the lowest of the n-quantiles being subjects with the lowest risk and the highest of the n-quantiles being subjects with the highest risk. In some embodiments, the predetermined level is a level or occurrence in the same subject, e.g., at a different time point, e.g., an earlier time point.

Subjects associated with predetermined values are typically referred to as reference subjects. For example, in some embodiments, a control reference subject does not have Angelman syndrome. A disease reference subject is one who has (or has an increased risk of developing) Angelman syndrome. An increased risk is defined as a risk above the risk of subjects in the general population.

Thus, in some cases the level of UBE3A in a subject being less than or equal to a reference level of UBE3A is indicative of Angelman syndrome. In other cases the level of UBE3A in a subject being greater than or equal to the reference level of UBE3A is indicative of the absence of Angelman syndrome or having low risk of developing Angelman syndrome. In cases where the level of UBE3A in a subject being equal to the reference level of UBE3A, the “being equal” refers to being approximately equal (e.g., not statistically different).

The predetermined value can depend upon the particular population of subjects selected. For example, an apparently healthy population will have a different ‘normal’ range of levels of UBE3A than will a population of subjects which have, are likely to have, or are at greater risk to have, Angelman syndrome. Accordingly, the predetermined values selected may take into account the category (e.g., sex, age, health, risk, presence of other diseases) in which a human subject falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art. In characterizing likelihood, or risk, numerous predetermined values can be established.

Additional methods of diagnosing a subject as having Angelman syndrome, or at risk for developing Angelman syndrome include genetic tests and behavioral symptom or physical appearance based diagnosis. For example, genetic tests such as chromosome analysis (e.g., fluorescent in situ hybridization (FISH) based chromosome analysis), DNA methylation test, and sequencing of the UBE3A gene are useful diagnostic tools. Behavioral symptoms or physical appearances for diagnosing Angelman syndrome include, but are not limited to, hand flapping or walking with arms in the air; jerky body movements; stiffed-leg walk; little or no speech; attention deficits; hyperactivity; feeding problems, e.g., in infancy; sleep problems and a need for less sleep than peers; delays in motor development; frequent laughter that may occur at inappropriate times; excitable personality; tongue thrusting; strabismus (crossing of the eyes); small head size with flatness in the back of the head; a lower jaw that juts out; light pigmentation in the hair skin and eyes. The methods of detecting presence or level of a UBE3 A protein in a subject’s sample can be used in combination with one or more of the diagnostic criteria described above for diagnosing a subject as having or at risk for developing Angelman syndrome.

Once it has been determined that a person has Angelman syndrome or has an increased risk of developing Angelman syndrome, then a treatment can be administered. Treatments for Angelman syndrome can include those that treat one or more symptoms related to Angelman syndrome. Suitable therapies include those for preventing or treating seizures (e.g. anticonvulsant medication); physical therapy (e.g. for maintaining or improving joint mobility and movement); speech therapy; occupational or behavioral therapy. Other treatments include those that increase neuronal UBE3A protein expression. For example, a treatment can be a gene therapy to increase activity of UBE3A protein, e.g., by increasing levels of functional UBE3A protein and/or increasing copies of a UBE3A gene that expresses functional protein. In other examples, a treatment can be a nucleic acid molecule, e.g., an oligonucleotide, e.g., an antisense oligonucleotide, that reduces the level of UBE3A antisense transcript (UBE3A-ATS), thereby increasing UBE3A protein production. The presence or level of UBE3A protein in a patient can be monitored during treatment by obtaining one or more samples (e.g., a CSF sample) from the patient at various time points and subject the samples to the methods of detection provided herein. In some embodiments, a subject diagnosed as having or at risk for developing Angelman syndrome (e.g., using any of the methods disclosed herein, alone or in combination) is administered a treatment for Angelman syndrome (e.g., any of the therapeutic treatments described herein, e.g., gene therapy or an antisense oligonucleotide). The level of UBE3 A protein in one or more samples obtained from the subject at one or more time points after initiation of treatment can be determined using the methods described herein. In some embodiments, a higher UBE3 A protein level after initiation of treatment compared to a control value (e.g., the range of UBE3A protein level found in subjects with Angelman syndrome) indicates that the therapeutic treatment is efficacious. In some embodiments, a higher UBE3A protein level after initiation of treatment compared to the UBE3A protein level in the subject prior to the initiation of treatment indicates that the therapeutic treatment is efficacious. In some embodiments, the treatment is deemed efficacious if progression of the disease is reduced or halted. In some embodiments, a higher UBE3 A protein level compared to a control value (or the UBE3A protein level measured in the subject prior to the initiation of treatment) indicates that the therapeutic treatment can be continued at the same or lower dosage and/or at the same or lengthened dosing interval. In some embodiments, a UBE3 A protein level equal to or lower than a control value (or the UBE3 A protein level measured in the subject prior to the initiation of treatment) indicates that the therapeutic treatment can be continued at a higher dosage and/or at a shortened dosing interval. Therapeutic treatments can continue until the UBE3A protein level in a subject is comparable to or higher than that of a control value (or higher than that of the UBE3A protein level measured in the subject prior to the initiation of treatment). In some embodiments, once the UBE3A protein level in a subject is comparable to or higher than that of a control value (or higher than that of the UBE3 A protein level measured in the subject prior to the initiation of treatment), maintenance doses of the therapeutic treatment can be administered thereafter. A “maintenance dose” refers to a constant dose level at which the therapeutic treatment is administered to the subject indefinitely, e.g., after the treatment is found to be efficacious in the subject. This disclosure also provides methods for determining whether a human subject is a candidate for a UBE3A targeted therapeutic. In one embodiment, the method entails identifying a subject as a candidate for a UBE3A targeted therapeutic by measuring according to a method described herein a UBE3 A protein level in a biological sample obtained from the human subject.

This disclosure also provides methods for identifying a human subject as having a lower or higher level of UBE3A protein than a reference level. In one embodiment, the method entails identifying a human subject as having a lower or higher level of UBE3A protein than a reference level by measuring according to a method described herein a UBE3A protein level in a biological sample obtained from the human subject and comparing the measured UBE3A protein level to a reference level.

This disclosure also provides methods for identifying a human subject as having or being at risk of developing a disease in which UBE3A protein levels are elevated compared to a reference. In one embodiment, the method entails identifying a human subject as having or being at risk of developing a disease in which UBE3A protein levels are elevated compared to a reference by measuring according to a method described herein a UBE3A protein level in a biological sample obtained from the human subject and comparing the measured UBE3A protein level to a reference, wherein the measured UBE3 A protein level is elevated compared to the reference.

This disclosure also provides methods for identifying a human subject as having or being at risk of developing a disease in which UBE3A protein levels are lower compared to a reference. In one embodiment, the method entails identifying a human subject as having or being at risk of developing a disease in which UBE3A protein levels are lower compared to a reference by measuring according to a method described herein a UBE3A protein level in a biological sample obtained from the human subject and comparing the measured UBE3A protein level to a reference, wherein the measured UBE3A protein level is lower compared to the reference.

In some methods described herein, e.g., where a subject is identified as having an elevated UBE3A protein level, a UBE3A targeted therapeutic is intended to reduce UBE3A protein levels or decrease EGBE3A activity. Examples of such therapeutics include an inhibitor of EGBE3A protein, an inhibitor of EGBE3A expression, transcription, or translation, or an inhibitor of a molecule in the same pathway as UBE3A. The therapeutic can be, for example, a nucleic acid (e.g., an antisense oligonucleotide that reduces the level of a UBE3 A transcript, thereby decreasing UBE3A protein production), a small molecule (e.g., a small molecule inhibitor of UBE3A activity), or an anti-UBE3A antibody (e.g., an antibody that binds to UBE3A and decreases activity of the protein). EXAMPLES

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art can develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Example 1: Immunoassays for UBE3A Detection

An immunoassay for UBE3 A detection based on the Singulex Errena system was developed. An exemplary protocol for the Singulex Errena system based assay is described in Fischer et al. AAPS J 17(1):93-101, 2015. EGBE3A standard protein (Abeam, abl25736) was used. The anti-UBE3A antibody GTX10488 (GeneTex) was used as the capture antibody, and the anti-UBE3A antibody H00007337-M02 (AbNova) was used as the detection antibody. Paramagnetic microparticles (MPs) were used as the solid phase for immune-capture and detection of the UBE3A standard protein in a microplate format. Signal generated by fluorescently labeled detection antibodies were counted as digital events, which corresponds to a single EGBE3A molecule. On average an MP was coated with 12.5 pg of the capture antibody, and the MPs were used at 10pg per well. The detection antibody was used at 500 ng/mL. Results from the assay showed high levels of background, as shown by the values in the “DE” column in Table 1 below. DE refers to all Digital Events recorded, which is used to calculate the Event Photons (EP). For reference, a typical background for this assay is between 50-100 DE.

Table 1

A further Singulex experiment was carried out using a different capture antibody, GTX101092 (GeneTex) and a different UBE3A standard protein (ab206157, Abeam). The same detection antibody, H00007337-M02 (AbNova) was used. On average an MP was coated with 25 pg of the capture antibody, and the MPs were used at 25pg per well. The detection antibody was used at 125 ng/mL. FIG. 1A shows a standard curve plotted based on the concentration of UBE3A and the DE values. FIG.

IB is an enlarged graph of the area marked in FIG. 1A. The standard curve was generated based on quadruplets. Table 2 shows the concentration of recombinant UBE3A protein that was loaded into the assay and respective recovery rate which was suitable over much of the observed range. Average DE ("AvgDE”) represents the raw signal from the assay. 21 CSF samples tested using this setup had a signal to noise ratio of less than 2, at both neat and 1:2 dilutions. UBE3A was detected in one brain tissue sample tested using this assay setup, however the signal to noise ratio was low, at about 2.5. Additional capture antibodies were also evaluated, however, none improved the performance of the assay.

Table 2

Next, a Quanterix Simoa assay system was used for UBE3 A detection. Exemplary protocols for the Quanterix Simoa system based assay are described in e.g., Fischer et al. AAPS J 17(1):93-101, 2015 and Wilson et al. Journal of Laboratory Automation, 21(4):533-547, 2016. The two-step Quanterix format was first evaluated to identify the optimal bead antibody and detector antibody combination. The assay condition used is shown in Table 3. Three antibodies, including GTX101092 (GeneTex, referred to as “GTX” herein after), 2F6 (WH0007337Ml-100ug, Millipore-Sigma) and NB500-240 (Novus, referred to as “Novus” herein after), were tested in pairs. The sensitivity of antibody combinations were assessed using a dilution curve of a recombinant EGBE3A protein. Table 4 shows results of the assessment (MS detector: Millipore-Sigma 2F6). AEB stands for the Average Enzymes per Bead and is the raw signal readout from the Quanterix Simoa assay system. The combination of GTX as the capture antibody, and Novus as the detection antibody lead to better sensitivity throughout the curve .

Table 3 Table 4

Further experiments were carried out to evaluate the three-step format of the Quanterix assay and identify an optimal pair of capture antibody and detection antibody. The assay conditions are shown in Table 5. Table 6 shows results of the assessment. Similar to the two-step format, the combination of GTX as the capture antibody, and Novus as the detection antibody was identified as the optimal pair.

Table 5

Table 6

Using the optimal pair of antibodies identified above, a comparison between the two-step format and the three-step format was carried out. Based on the results (Table 7), the three-step format has a lower background and better dynamic range, but the two- step format has higher sensitivity, and so the two-step format was selected for further evaluation. Table 7

An additional anti-UBE3A antibody 3E5 (SAB 1404508- lOOug, Millipore- Sigma) was evaluated either as a capture antibody or a detector antibody, in combination with GTX, Novus, or MS 2F6, using both the two-step and three-step formats. The same conditions as those shown in Tables 3 and 5 were used. As shown in Table 8, GTX as the capture antibody and the MS 3E5 as the detector antibody showed a strong signal throughout the curve; however, MS 2F6 as the capture antibody and MS 3E5 as the detector antibody showed better signal to background ratio for lower end of the curve. Similar results were obtained from the two-step format (Table 9). Higher AEBs were observed for the combination of GTX as the capture antibody and MS 3E5 as the detector antibody at 1 ng/mL and 10 ng/mL UBE3a levels, when compared to other combinations. However, MS 2F6 as the capture antibody and MS 3E5 as the detector antibody showed better signal to background ratio for lower end of the curve.

Table 8

Table 9

A head-to-head comparison of the top performing antibody pairs in both the two-step format and the three-step format was carried out. These antibody pairs were tested against a dilution curve of recombinant UBE3A. As shown in Tables 10 and 11, the combination of GTX as the capture antibody and MS 3E5 as the detector antibody under the three-step format was identified as the optimal assay condition with the lowest assay LOD and highest sensitivity.

Table 10

Table 11

Next an initial run was carried out, using the 3 -step format and the GTX/MS 3E5 combination. The recombinant UBE3A solution was incubated with the GTX- conjugated beads for 120 minutes, which is followed by 5 minutes of incubation with the detector antibody, and 5 minutes of incubation with SBG. Results are shown in Table 12.

Table 12

Further optimization of the protocol included modifying the concentration of the capture antibody 3E5 (Table 13). O.lpg/mL of 3E5 was selected. Table 14 shows results from modifications of streptavidin b-galactosidase (SBG) concentrations. 100pm SBG was selected.

Table 13

Addition of helper beads and varying 3E5 levels were tested in combination, and no helper beads were selected (Tables 15 ar

16). Table 15

Table 16

Next, CSF samples were subjected to various detergent conditions, including 0.5% Tween, 1% Triton X-100, 0.5% Triton X-100, 1% SDS, 0.5% SDS, and 0.1%

SDS (FIG. 2). Ionic detergents such as SDS was shown to not be compatible with the assay. 1% Triton X-100 showed improved performance, with 3 of the 5 samples above limit of detection (LOD; 0.0308 ng/mL), and 2 above lower limit of quantitation (LLOQ; 0.118 ng/mL). 1% Triton X-100 was therefore selected as the optimal detergent condition.

With the optimized antibody pair and assay conditions, 10 CSF samples were tested. 40 pL of CSF from 10 independent donors were ran in triplicates. The results (FIG. 3) showed that all 10 samples were below the LLOQ of the assay. Therefore, it was concluded that the assay was not suitable for detection of UBE3A in human CSF samples.

Example 2: LC-MS Based Detection of UBE3A

A liquid chromatography-mass spectrometry (LC-MS) protocol was developed for detecting UBE3A.

Sigma anti-UBE3A monoclonal antibody (clone 3E5, cat. SAB1404508-100ug) was biotinylated using a Thermo Fisher biotinylation kit and then labeled onto M280 Streptavidin coated magnetic beads. 500 pL sample (including 500 pL CSF, or recombinant UBE3A) were placed in Eppendorf LoBind tubes. 50 pL of 10X RIPA lysis buffer (EMD Millipore cat. 20-188) was added to each sample. 10 pL of antibody- conjugated beads were added to each sample. Samples were incubated with beads on end over end mixer (HulaMixer) for 2 hours at 4°C. Samples were placed on a tube magnet and liquid was discarded. 1 mL PBS + Tween-20 (0.01%) was added to each tube and then mixed on an end over end mixer (Hulamixer) at room temperature for 1 minute. Samples were put on a magnet and liquid was discarded. 1 mL PBS was added to each tube and then mixed on an end over end mixer (Hulamixer) at room temperature for 1 minute. Samples were put on a magnet and liquid was discarded. 0.5 mL PBS was added to each tube and then mixed on an end over end mixer (Hulamixer) at room temperature for 1 minute. Samples were put on a magnet and liquid was discarded. TPCK-treated trypsin (Worthington cat. LS003740) was made up to 1 mg/mL in PBS. Trypsin was then diluted by adding 3 pL /450 pL of PBS to make the digestion solution. 45 pL of the digestion solution was added to each tube with beads. Samples were placed on ThermoMixer at 40 °C for 1.5 hrs. 5 pL of 20% acetonitrile and 1% formic acid were added to each sample. Samples were put on a magnet and final solution (50 pL) was added to plate for LC-MS analysis. Detectable peptides in the human UBE3A protein, and the relative abundance of these peptides can be found in Table 17. The detectable peptides are listed in the column “sequence” and the relative abundance can be found in the “area” column. “A2” refers to “detection confidence”, and represents the confidence of the mass spectrometry system for detection of the particular peptide.

Table 17

The most abundant detectable peptides included: VFSSAEALVQSFR (SEQ ID N0:3), NLVNDDDAIVAASK (SEQ ID N0:4), VDPLETELGVK (SEQ ID N0:5), and LEMIAMENPADLKK (SEQ ID N0:6). These peptides are bolded in the sequence of UBE3A below: MEKLHQCYWKSGEPQSDDIEASRMKRAAAKHLIERYYHQLTEGCGNEACTNEFCASCP TFLRMDNNAAAIKALELYKINAKLCDPHPSKKGASSAYLENSKGAPNNSCSEIKMNKK GARIDFKDVTYLTEEKVYEILELCREREDYSPLIRVIGRVFSSAEALVQSFRKVKQHT KEELKSLQAKDEDKDEDEKEKAACSAAAMEEDSEASSSRIGDSSQGDNNLQKLGPDDV SVDIDAIRRVYTRLLSNEKIETAFLNALVYLSPNVECDLTYHNVYSRDPNYLNLFIIV MENRNLHSPEYLEMALPLFCKAMSKLPLAAQGKLIRLWSKYNADQIRRMMETFQQLIT

YKVISNEFNSRNLVNDDDAIVAASKCLKMVYYANW GGEVDTNHNEEDDEEPIPESSE LTLQELLGEERRNKKGPRVDPLETELGVKTLDCRKPLIPFEEFINEPLNEVLEMDKDY TFFKVETENKFSFMTCPFILNAVTKNLGLYYDNRIRMYSERRITVLYSLVQGQQLNPY LRLKVRRDHIIDDALVRLEMIAMENPADLKKQLYVEFEGEQGVDEGGVSKEFFQLW E EIFNPDIGMFTYDESTKLFWFNPSSFETEGQFTLIGIVLGLAIYNNCILDVHFPMW Y

RKLMGKKGTFRDLGDSHPVLYQSLKDLLEYEGNVEDDMMITFQISQTDLFGNPMMYD L KENGDKIPITNENRKEFVNLYSDYILNKSVEKQFKAFRRGFHMVTNESPLKYLFRPEE IELLICGSRNLDFQALEETTEYDGGYTRDSVLIREFWEIVHSFTDEQKRLFLQFTTGT DRAPVGGLGKLKMIIAKNGPDTERLPTSHTCFNVLLLPEYSSKEKLKERLLKAITYAK GFGML (SEQ ID NO:1)

CSF samples from 14 independent donors were collected and subjected to the above protocol. In 12 out of the 14 samples, UBE3A was detected (FIG. 4). The assay was also highly reproducible. Pooled CSF was split into 6 aliquots of 500 pL each and subjected to the above protocol with 5% CV in 6 replicates ran in parallel (FIG. 5).

To further evaluate assay specificity, UBE3A in HEK293T cells was knocked down by UBE3A shRNA prior to running the detection assay. Specifically, HEK293T cells were either treated with control shRNA or 1 of 4 UBE3 A shRNAs. Cells were lysed and the LC-MS assay described above was performed on these lysates. As shown in FIG. 6, a reduction in the UBE3 A signals were observed in cells where UBE3 A was knocked down.

To see if the UBE3 A signal detected using the assay correlated with the CSF volume, different volumes of CSF (1 mL, 500 pL, 250 pL, 125 pL, 0 pL) from a single source were tested. As shown in FIGs. 7A and 7B, good parallelism between the mass spectrometry signal and the volume of CSF samples were observed. These results demonstrate that the LC-MS assay allows specific detection of the UBE3A protein in patient CSF samples. The detection was selective, based on recombinant linearity, endogenous parallelism, and tissue KD.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.