Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/04—Preparation or injection of sample to be analysed
  • G01N30/06—Preparation
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
  • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
  • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/62—Detectors specially adapted therefor
  • G01N30/72—Mass spectrometers

Definitions

• the present application hereby incorporates by reference the entire contents of the XML file named “206017-022 l-OOWO_SequenceListing.xml” in XML format, which was created on August 16, 2023, and is 295,220 bytes in size.
• MMC Myelomeningocele
• NTD congenital open neural tube defect
• the presentation is commonly in the lumbosacral region, and the defect is characterized by protrusion of the malformed spinal cord and meninges through a pathological opening in the overlying vertebrae and skin, leaving the spinal cord exposed to the intrauterine environment (Kaufman, B. A., 2004, Pediatiric Clinics of North America, 51 :389-419).
• the underlying defect leads to prenatal injury to the exposed spinal cord and a spectrum of associated abnormalities resulting in life-long disability including leg paralysis, sensory loss, bowel and bladder dysfunctions, skeletal deformations, Arnold-Chiari type TI malformation, hindbrain herniation and development of hydrocephalus (Hunt, G.
• AFP alpha fetoprotein
• AF amniotic fluid
• the present invention relates to a method of diagnosing an open Neural Tube Defect (NTD) in a human fetus.
• NTD Neural Tube Defect
• the invention relates to a method of diagnosing open NTD comprising the steps of obtaining a sample of amniotic fluid and/or other bodily fluids from a fetus or an individual carrying the fetus; the detecting of the levels or differential patterns of neurocan and/or RPTPp/phosphacan and comparing the levels and/or differential patterns of neurocan and/or RPTPp/phosphacan with that of a normal sample.
• the detecting of the levels and/or differential pattern of neurocan and/or RPTPp/phosphacan further comprises the step of treating the biological sample with a chondroitinase or any enzyme able to remove and/or modify glycosaminoglycan (GAG) component.
• GAG glycosaminoglycan
• the detecting of the levels and/or differential pattern of neurocan and/or RPTPp/phosphacan does not include the step of removal and/or modification of GAG component.
• the detecting of the levels and/or differential pattern of neurocan and/or RPTPp/phosphacan further comprises detecting the levels and/or differential pattern of at least one member chosen from the group consisting of the glycosaminoglycan (GAG)-containing and/or GAG-lacking neurocan proteins (encoding gene ID number : 1463, neurocan, SEQ ID NO: 1), a full length neurocan protein, the proteolytically cleaved N-terminal neurocan fragment, the proteolytically cleaved C-terminal neurocan fragment, and/or other products of their fragmentation, the GAG-containing and/or GAG lacking RPTPp/phosphacan protein isoforms (encoding gene ID number: 5803, protein tyrosine phosphatase receptor type Zl, also known as RPTPp, phosphacan, SEQ ID NO:2), full-length RPTPp/phosphacan protein isoforms and/or products of their fragmentation.
• GAG glyco
• the detecting of the levels and/or differential pattern of neurocan and/or RPTPp/phosphacan involves the use of an antibody to at least one molecular form of neurocan and/or RPTPp/phosphacan.
• the detecting of the levels and/or differential pattern of neurocan and/or RPTPp/phosphacan is performed without the use of an antibody.
• the detecting of the levels and/or differential pattern of neurocan and/or RPTPp/phosphacan is accomplished via Western blotting and immunoblotting.
• the detecting of the levels of neurocan and/or RPTPp/phosphacan is accomplished by employing an ELISA assay.
• the detecting of the levels and/or differential pattern of neurocan and/or RPTPp/phosphacan is accomplished by employing any biological or chemical assay that specifically detects at least one molecular form of neurocan and/or RPTPp/phosphacan or any component of these molecules.
• Figure 1 depicts retinoic acid-induced myelomeningocele (MMC) in fetal rats and the experimental strategy employed.
• Figure 1A depicts a representative external view of RA-induced MMC defect in the lumbar region of a fetal rat at embryonic day 21 (E21). The arrow indicates the beginning of the exposed spinal cord.
• Figure IB depicts representative imaging of a hematoxylin and eosin (H&E) stained cross section from the MMC defect in a fetal rat at E21, demonstrating the malformed spinal cord and failed development of overlying structures. Scale bar indicates 100 pm.
• Figure 1C depicts a schematic representation of an experimental strategy employed.
• Figure 2 depicts schematic illustration of neurocan and RPTPp/phosphacan and the representative detection of neurocan and phosphacan in the amniotic fluid of MMC fetuses.
• Figure 2A depicts schematic representation of the domain organization of neurocan and its major proteolytic fragments, N-terminal fragment, and C-terminal fragment, and a long splice form of RPTPp and its extracellular variant, phosphacan. Domains recognized by 1F6 and 650.24 or 3F8 antibodies are indicated.
• Figure 2B depicts a representative Western blot performed with equal volumes of amniotic fluid samples pooled from three randomly selected MMC fetuses or age-matched normal fetuses collected at E14, E16, E18, and E21 after being digested with ChABC.
• the western blot analysis depicted was performed with 1F6 antibody, illustrating detection of full-length neurocan core protein and the proteolytically cleaved N-terminal fragment (245 kDa and 130 kDa), respectively and 650.24 antibody, illustrating detection of full-length neurocan core protein and the proteolytically cleaved C-terminal fragment (245 kDa and 150 kDa), respectively.
• FIG. 2C depicts quantitative analysis of total levels of neurocan proteins in the AF of MMC fetuses at all embryonic ages examined using 1F6 antibody as depicted in Figure 2B.
• Figure 2D depicts quantitative analysis of total levels of neurocan proteins in the AF of MMC fetuses and age-matched normal controls examined using 650.24 antibody as depicted in Figure 2B.
• Figure 2E depicts representative quantitative analysis of total levels of phosphacan protein in the AF of MMC fetuses and age-matched normal controls examined using 3F8 antibody as depicted in Figure 2B . All graphs represent the change in arbitrary fluorescence units (AFU) at each time point. Data are presented as mean ⁇ SD, of 15 randomly selected AF samples per group. *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001, ****, p ⁇ 0.0001.
• Figure 2F depicts a representative western blot of neurocan and phosphacan illustrating their detection in all AF samples from individual E21 fetuses with MMC defect, but very weak or no detection in the AF of fetuses with no MMC defect after RA exposure or in normal controls. Data represents 6 AF samples per group.
• Figure 3 depicts extractions of phosphacan and neurocan from MMC spinal cord tissues.
• Figure 3A depicts a representative Western blot analysis of the first (extracted without detergent) and second (extracted with detergent) sequential extracts of spinal cords isolated from MMC fetuses at E14, E16, E18, and E21 that were ChABC-digested and subjected to Western blot analysis using 1F6, 650.24, or 3F8 antibodies, demonstrating the vast majority of neurocan and phosphacan detected in the first extract at all gestational ages.
• Figure 3B depicts a representative Western blot analysis of AF samples from E21 MMC fetuses, AF samples from E21 normal controls, and AF samples from normal E21 controls that were incubated with spinal cords isolated from E21 MMC fetuses and then subjected to Western blot analysis using 1F6, 650.24, or 3F8 antibodies, demonstrating their release from MMC spinal cord tissue into the AF.
• Data represent spinal cord extracts from at least two independent sets of spinal cord tissue samples per group.
• Figure 4 depicts representative images of neurocan and phosphacan expression in MMC spinal cord tissues and its immunohistochemical characterization.
• Figure 4A depicts representative micrographs illustrating localization of RPTPfi/phosphacan (white) and Neurocan (yellow) using RNAscope in MMC spinal cords examined at El 4 (left panel) and E21 (right panel). Cell nuclei were fluorescently stained with DAPI (blue).
• Figure 4B depicts the type of cells that synthetize RPTPfi/phosphacan and Neurocan identified by coimmunostaining for glutamine synthetase (GS; green), a marker of astrocytic cells; MAP2 (magenta), a marker of neurons; and RNAscope for RPTPfPphosphacan (white); or Neurocan (yellow) in spinal cord sections from MMC defects examined at E21.
• Figure 4C depicts representative micrographs illustrating extracellular distribution pattern of phosphacan and neurocan in the immunostained spinal cord sections obtained through the center of MMC defect examined at E21. Cell nuclei were fluorescently stained with DAPI (blue). Scale bar represents 100pm. Data represent at least three sections from three fetuses per group.
• the present invention is based in part on the discovery that elevated levels of neurocan and/or RPTPp/phosphacan is an effective predictor of an open NTD.
• the present invention relates to methods relating to biomarkers (e.g., one or more chondroitin sulfate proteoglycans (CSPGs), neurocan and/or RPTPp/phosphacan) that can be used for identifying and diagnosing open NTD in human fetuses.
• biomarkers e.g., one or more chondroitin sulfate proteoglycans (CSPGs), neurocan and/or RPTPp/phosphacan
• CSPGs such as neurocan and RPTPp/phosphacan are molecules consisting of a protein core with the attached glycosaminoglycan (GAG) component.
• GAG glycosaminoglycan
• biological sample obtained from the subject can be analyzed before and after GAG removal.
• the diagnosis is accomplished by comparison of levels and/or differential patterns of GAG- containing and/or GAG-lacking CSPGs.
• the CSPGs are at least one selected from the group consisting of neurocan, RPTPp/phosphacan, brevican, aggrecan, versican.
• the diagnosis is accomplished by comparison of levels and/or differential patterns of GAG-containing and/or GAG-lacking neurocan and/or RPTPp/phosphacan proteins in a sample of amniotic fluid or other bodily fluids obtained from a fetus or an individual carrying the fetus.
• the detecting of the levels of neurocan and/or RPTPp/phosphacan comprises detection of total GAG-containing and/or GAG lacking neurocan and/or RPTPp/phosphacan proteins.
• detecting of the levels and/or differential pattern of neurocan and/or RPTPp/phosphacan comprises detection of individual GAG-containing and/or GAG lacking full-length neurocan and/or full length RPTPp/phosphacan protein isoforms, the proteolytically cleaved N-term in al neurocan fragment, the proteolytically cleaved C-terminal neurocan fragment, and/or any other neurocan, and/or RPTPp/phosphacan fragmentation products, or any combination of thereof.
• detecting localization of neurocan and/or RPTPp/phosphacan comprises detection of colocalization of neurocan and/or RPTPp/phosphacan with markers of astrocytic cells and/or neurons and/or detection of the extracellular distribution patterns of neurocan and/or RPTPp/phosphacan.
• the step of analyzing the biological sample further comprises a step of treating the biological sample with chondroitinase ABC or any enzyme able to remove and/or modify GAG component. In one embodiment, the analysis of the biological sample does not include the step of removing and/or modifying GAG component.
• the detecting of the levels and/or differential pattern of neurocan and/or RPTPp/phosphacan involves the use of an antibody to at least one molecular form of neurocan and/or RPTPp/phosphacan. In one embodiment, the detecting of the levels and/or differential pattern of neurocan and/or RPTPp/phosphacan is performed without the use of an antibody. In one embodiment, the diagnosis involves the use of Western blotting and immunoblotting.
• the diagnosis involves employing an ELISA assay, or any other chemical or biological assay e.g., HPLC that detects at least one molecular form of neurocan and/or RPTPp/phosphacan, their proteolytically cleaved fragments, and/or other fragmentation products.
• the detecting of the colocalization or extracellular distribution pattern of neurocan and/or RPTPp/phosphacan comprises RNAscope and/or coimmunostaining.
• antibody refers to an immunoglobulin molecule which is able to specifically bind to a specific epitope of an antigen.
• Antibodies can be intact immunoglobulins derived from natural sources, or from recombinant sources and can be immunoreactive portions of intact immunoglobulins.
• the antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“intrabodies”), Fv, Fab, Fab’, F(ab)2 and F(ab’)2, as well as single chain antibodies (scFv), heavy chain antibodies, such as camelid antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).
• synthetic antibody as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage.
• the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
• the term “heavy chain antibody” or “heavy chain antibodies” comprises immunoglobulin molecules derived from camelid species, either by immunization with a peptide and subsequent isolation of sera, or by the cloning and expression of nucleic acid sequences encoding such antibodies.
• the term “heavy chain antibody” or “heavy chain antibodies” further encompasses immunoglobulin molecules isolated from a subject with heavy chain disease, or prepared by the cloning and expression of VH (variable heavy chain immunoglobulin) genes from a subject.
• a “chimeric antibody” refers to a type of engineered antibody which contains a naturally-occurring variable region (light chain and heavy chains) derived from a donor antibody in association with light and heavy chain constant regions derived from an acceptor antibody.
• a “humanized antibody” refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s).
• framework support residues may be altered to preserve binding affinity (see, e.g., 1989, Queen et al., Proc. Natl. Acad Sci USA, 86: 10029-10032; 1991, Hodgson et al., Bio/Technology, 9:421).
• a suitable human acceptor antibody may be one selected from a conventional database, e.g., the KABAT database, Los Alamos database, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody.
• a human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and/or a heavy chain variable framework region for insertion of the donor CDRs.
• a suitable acceptor antibody capable of donating light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody.
• the prior art describes several ways of producing such humanized antibodies (see for example EP-A-0239400 and 20 EP-A-054951).
• donor antibody refers to an antibody (monoclonal, and/or recombinant) which contributes the amino acid sequences of its variable regions, CDRs, or other functional fragments or analogs thereof to a first immunoglobulin partner, so as to provide the altered immunoglobulin coding region and resulting expressed altered antibody with the antigenic specificity and neutralizing activity characteristic of the donor antibody.
• acceptor antibody refers to an antibody (monoclonal and/or recombinant) heterologous to the donor antibody, which contributes all (or any portion, but in some embodiments all) of the amino acid sequences encoding its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to the first immunoglobulin partner.
• a human antibody is the acceptor antibody.
• assessing includes any form of measurement, and includes determining if an element is present or not.
• the terms “determining,” “measuring,” “evaluating,” “assessing” and “assaying” are used interchangeably and may include quantitative and/or qualitative determinations. Assessing may be relative or absolute.
• “Assessing binding” includes determining the amount of binding, and/or determining whether binding has occurred (i.e., whether binding is present or absent). “Assessing activity” includes determining the amount of activity, and/or determining whether an activity has occurred (i.e., whether an activity is present or absent).
• binding refers to a direct association between at least two molecules, due to, for example, covalent, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions.
• biological sample is intended to include any sample comprising a cell, a tissue, or a bodily fluid in which expression of a nucleic acid or polypeptide can be detected.
• the biological sample may contain any biological material suitable for detecting the desired biomarkers, and may comprise cellular and/or non-cellular material obtained from the individual. Examples of such biological samples include but are not limited to blood, lymph, bone marrow, biopsies and smears.
• Bio fluids Samples that are liquid in nature are 5 referred to herein as “bodily fluids.”
• Biological samples may be obtained from a patient by a variety of techniques including, for example, by scraping or swabbing an area or by using a needle to obtain bodily fluids. Methods for collecting various body samples are well known in the art.
• diagnosis and “diagnosing” refer to the determination of the presence of a disease or disorder.
• methods for making a diagnosis are provided which permit determination of the presence of a myelomeningocele.
• epitope refers to the specific group of atoms on an antigen molecule to which a specific antibody binds, causing an immune response.
• an “immunoassay” refers to any binding assay that uses an antibody capable of binding specifically to a target molecule to detect and quantify the target molecule.
• a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
• the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
• label when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to a probe to generate a “labeled” probe.
• the label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable (e.g., avidin-biotin).
• primers can be labeled to detect a PCR product.
• an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample.
• an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific.
• an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.
• the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.
• a particular structure e.g., an antigenic determinant or epitope
• the terms “specific binding” or “specifically binding”, can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.
• a particular structure e.g., an antigenic determinant or epitope
• Measurement or “measurement,” or alternatively “detecting” or “detection,” means assessing the presence, absence, quantity or amount (which can be an effective amount) of either a given substance within a sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise evaluating the values or categorization of the substance or the sample.
• nucleic acid refers to a polynucleotide and includes poly-ribonucleotides and poly-deoxyribonucleotides.
• Nucleic acids according to the present invention may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively. (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982) which is herein incorporated in its entirety for all purposes).
• the present invention contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glycosylated forms of these bases, and the like.
• the polymers or oligomers may be heterogeneous or homogeneous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced.
• the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in singlestranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.
• patient refers to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein.
• the patient, subject, or individual is a human.
• ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
• the present invention is based, in part, on the discovery that various proteins are elevated in the amniotic fluid of fetuses with an open NTD. Accordingly, in various embodiments the invention is directed towards diagnosing open NTD by detecting the levels of neurocan and/or RPTPp/phosphacan in amniotic fluid or other bodily fluids.
• the diagnosis is accomplished by comparison of levels and/or differential patterns of GAG-containing and/or GAG-lacking CSPGs.
• the CSPGs are at least one selected from the group consisting of neurocan, RPTPp/phosphacan, brevican, aggrecan, versican.
• the invention is directed towards the detection of the levels of total GAG-containing and/or GAG-lacking neurocan proteins.
• the invention is directed towards the detection of the levels and/or differential patterns of individual GAG-containing and/or GAG-lacking full length neurocan protein, the proteolytically cleaved N-terminal neurocan fragment, the proteolytically cleaved C-terminal neurocan fragment, and/or other products of neurocan fragmentation, or any combination of thereof.
• the invention is directed towards the detection of the levels of total GAG-containing and/or GAG-lacking RPTPp/phosphacan proteins.
• the invention is directed towards the detection of the levels and/or differential patterns of individual GAG-containing and/or GAG-lacking full length RPTPp/phosphacan proteins, and/or products of RPTPp/phosphacan fragmentation, or any combination of thereof.
• the present invention provides methods for diagnosing open NTD in a subject in need thereof. In one aspect, the present invention also provides methods for distinguishing a subject with open NTD from a subject without open NTD.
• the method comprises obtaining a biological sample from a test subject. In one embodiment, the method comprises analyzing the biological sample with an assay that specifically detects at least one molecular form of one neurocan and/or RPTPp/phosphacan. In some embodiments, the method comprises detecting the level of at least one molecular form of neurocan and/or RPTPp/phosphacan in the biological sample of the subject. In various embodiments, the method comprises comparing the level of at least one molecular form of neurocan and/or RPTPp/phosphacan to a comparator. In some embodiments, the method comprises determining that the subject has open NTD.
• the method comprises at least one molecular form of neurocan and/or RPTPp/phosphacan. In one embodiment, the method comprises two or more molecular forms of neurocan and/or RPTPp/phosphacan. In one embodiment, the method comprises three or more molecular forms of neurocan and/or RPTPp/phosphacan. In one embodiment, the method comprises four or more molecular forms of neurocan and/or RPTPp/phosphacan.
• the method comprises the detecting of (GAG)- containing and/or GAG-lacking neurocan proteins (encoding gene ID number : 1463, neurocan, SEQ ID NO: 1), a full length neurocan protein, the proteolytically cleaved N-terminal neurocan fragment, the proteolytically cleaved C-terminal neurocan fragment, and/or other products of neurocan fragmentation.
• GAG GAG-containing and/or GAG-lacking neurocan proteins
• the method comprises the detecting of the GAG- containing and/or GAG lacking RPTPp/phosphacan protein isoforms (encoding gene ID number: 5803, protein tyrosine phosphatase receptor type Z1 gene, also known as RPTPp, phosphacan, SEQ ID NO:2), full-length RPTPp/phosphacan protein isoforms and/or products of their fragmentation.
• RPTPp/phosphacan protein isoforms encoding gene ID number: 5803, protein tyrosine phosphatase receptor type Z1 gene, also known as RPTPp, phosphacan, SEQ ID NO:2
• the method for diagnosing open NTD comprises detecting the levels of total GAG-containing and/or GAG lacking neurocan and/or RPTPp/phosphacan proteins.
• detecting of the levels and/or differential pattern of neurocan and/or RPTPp/phosphacan comprises detection of individual GAG- containing and/or GAG lacking full-length neurocan and/or RPTPp/phosphacan proteins, the proteolytically cleaved N-terminal neurocan fragment, the proteolytically cleaved C-terminal neurocan fragment, and/or any other neurocan and/or RPTPp/phosphacan fragmentation products, or any combination thereof.
• the method of detecting the levels of neurocan and/or RPTPp/phosphacan comprises the additional step of treating the sample with a chondroitinase, or chondroitin lyase.
• a chondroitinase or chondroitin lyase.
• treating the sample with a chondroitinase while catalytically cleaving the polysaccharide post-translational modifications of neurocan and/or RPTPp/phosphacan, will not diminish one’s ability to accurately detect protein levels.
• any number of chondroitinases may be employed, including, but not limited to, chondroitinase AC, chondroitinase ABC, and chondroitinase B.
• the detecting of the levels and/or differential pattern of neurocan and/or RPTPp/phosphacan does not include the step of removal and/or modification of GAG component.
• the method comprises determining if the level of the relevant neurocan and/or RPTPp/ phosphacan is elevated as compared to a comparator.
• the comparator may be the level (e g., amount, concentration, concentration of one or more molecular forms, concentration of the neutral form, expression, level, etc.) of neurocan and/or RPTPp/phosphacan in a subject without open NTD.
• the comparator is the level (e.g., amount, concentration, concentration of one or more forms, concentration of the neutral form, expression, level, etc.) of neurocan and/or RPTPp/phosphacan obtained from a human subject, an average of multiple human subjects, an average of multiple human subjects living in the same region, an average of human subjects with the same race, an average of multiple human subjects with the same ethnicity, an average of multiple human subjects living in different regions, an average of multiple human subjects with different races, an average of multiple human subjects with different ethnicities, or any combination thereof.
• a human subject an average of multiple human subjects, an average of multiple human subjects living in the same region, an average of human subjects with the same race, an average of multiple human subjects with the same ethnicity, an average of multiple human subjects living in different regions, an average of multiple human subjects with different races, an average of multiple human subjects with different ethnicities, or any combination thereof.
• the method comprises detecting at least one selected from the group consisting of the levels, ratios, and differential patterns of at least one neurocan and/or RPTPp/phosphacan in a biological sample obtained from the subject, wherein at least one member is chosen from the group consisting at least one selected from the group consisting of GAG-containing full length neurocan protein (encoding gene ID number : 1463, neurocan, SEQ ID NO:1), GAG-lacking full length neurocan protein, GAG-containing full-length RPTPp/phosphacan protein isoform (encoding gene ID number: 5803, protein tyrosine phosphatase receptor type Z1 gene, also known as RPTPp, phosphacan, SEQ ID NO: 2), GAG- lacking full-length RPTPp/phosphacan protein isoform, GAG-containing proteolytically cleaved N-terminal neurocan fragment, GAG-lacking proteolytically cleaved N-terminal neurocan fragment, GAG-containing prote
• a subject is identified as having open NTD when the level of total GAG-containing and/or GAG-lacking neurocan proteins (encoding gene ID number : 1463, neurocan, SEQ ID NO: 1), is increased in the biological sample as compared to the comparator.
• a subject is identified as having open NTD when a ratio of total GAG-containing and/or GAG-lacking neurocan proteins is increased in the biological sample as compared to the comparator.
• a subject is identified as having open NTD when the level of total GAG-containing and/or GAG-lacking RPTPp/phosphacan proteins (encoding gene ID number:5803, protein tyrosine phosphatase receptor type Zl gene, also known as RPTPP, phosphacan, SEQ ID NO:2) is increased in the biological sample as compared to the comparator.
• RPTPp/phosphacan proteins encoding gene ID number:5803, protein tyrosine phosphatase receptor type Zl gene, also known as RPTPP, phosphacan, SEQ ID NO:2
• a subject is identified as having open NTD when the ratio of total GAG-containing and/or GAG-lacking RPTPp/phosphacan proteins is increased in the biological sample as compared to the comparator.
• a subject is identified as having open NTD when the level of proteolytically cleaved N-terminal neurocan fragment, is increased in the biological sample as compared to the comparator.
• a subject is identified as having open NTD when the level of proteolytically cleaved C-terminal neurocan fragment, is increased in the biological sample as compared to the comparator.
• a subject is identified as having open NTD when the level and/ or pattern of neurocan fragmentation products is increased and/or changed in the biological sample as compared to the comparator.
• a subject is identified as having open NTD when the level of any protein isoform of RPTPp/phosphacan (encoding gene ID number:5803 protein tyrosine phosphatase receptor type Z1 gene, also known as RPTPP, phosphacan, SEQ ID NO:2) is increased in the biological sample as compared to the comparator.
• RPTPp/phosphacan encoding gene ID number:5803 protein tyrosine phosphatase receptor type Z1 gene, also known as RPTPP, phosphacan, SEQ ID NO:2
• a subject is identified as having open NTD when the level and/ or pattern of at least one RPTPp/phosphacan fragmentation product is increased and/or changed in the biological sample as compared to the comparator.
• the level (e.g., activity, amount, concentration, concentration of one or more ionized forms, concentration of the neutral form, expression, level, etc.) of at least one molecular form of neurocan and/or RPTPp/phosphacan, or any combination thereof is determined to be increased when the level of at least one molecular form of neurocan and/or RPTPp/phosphacan, or any combination thereof, in the biological sample is increased by at least 0.01 fold, at least 0.05 fold, at least 0.07 fold, at least 0.076 fold, at least 0.1 fold, at least 0.18 fold, at least 0.19 fold, at least 0.3 fold, at least 0.36 fold, at least 0.37 fold, at least 0.38 fold, at least 0.4 fold, at least 0.43 fold, at least 1 fold, at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least
• the level (e.g., activity, amount, concentration, concentration of one or more ionized fonns, concentration of the neutral form, expression, level, etc.) of at least one molecular form of neurocan and/or RPTPp/phosphacan, or any combination thereof is determined to be increased when the level of at least one molecular form of neurocan and/or RPTPp/phosphacan, or any combination thereof, in the biological sample is increased by at least 1%, by at least 5%, by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 125%, by at least 150%, by at least 175%, by at least 200%, by at least 250%, by at least 300%, by at least 400%, by at least 500%, by at least 600%, by at least 700%, by at least 800%, by
• the ratio of neurocan to a known biomarker of spinal cord injury in the amniotic fluid is determined to be increased when the ratio is increased by at least 1%, by at least 5%, by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 125%, by at least 150%, by at least 175%, by at least 200%, by at least 250%, by at least 300%, by at least 400%, by at least 500%, by at least 600%, by at least 700%, by at least 800%, by at least 900%, by at least 1000%, by at least 1500%, by at least 2000%, by at least 2500%, by at least 3000%, by at least 4000%, or by at least 5000%, when compared to a comparator.
• the ratio of phosphacan to a known biomarker of spinal cord injury in the amniotic fluid is determined to be increased when the ratio is increased by at least 1%, by at least 5%, by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 125%, by at least 150%, by at least 175%, by at least 200%, by at least 250%, by at least 300%, by at least 400%, by at least 500%, by at least 600%, by at least 700%, by at least 800%, by at least 900%, by at least 1000%, by at least 1500%, by at least 2000%, by at least 2500%, by at least 3000%, by at least 4000%, or by at least 5000%, when compared to a comparator.
• methods of measuring neurocan and/or RPTPp/ phosphacan levels in a biological sample obtained from a subject include, but are not limited to, an immunochromatography assay, an immunodot assay, a Luminex assay, an ELISPOT assay, a protein microarray assay, a ligand-receptor binding assay, displacement of a ligand from a receptor assay, displacement of a ligand from a shared receptor assay, an immunostaining assay, an RNAscope assay, a radioimmunoassay (RIA), a radioimmunodiffusion assay, an Ouchterlony immunodiffusion assay, reverse phase protein microarray, a rocket immunoelectrophoresis assay, an immunohistostaining assay, an immunoprecipitation assay, a complement fixation assay, an enzyme-substrate binding assay, an enzymatic assay, an enzymatic assay employ
• the concentration of the neurocan and/or RPTPp/phosphacan or their fragmentation products in a sample may be determined by any suitable assay.
• a suitable assay may include one or more of the following methods, an enzyme assay, an immunoassay, mass spectrometry, chromatography, electrophoresis or an antibody microarray, or any combination thereof.
• the systems and methods of the invention may include any method known in the art to detect a protein in a sample.
• the method of detecting the levels and/or differential patterns of neurocan and/or RPTPp/phosphacan is accomplished by Western blotting followed by immunoblotting with an antibody to at least one molecular form of neurocan and/or RPTPp/phosphacan.
• the method of detecting colocalization of neurocan and/or RPTPp/phosphacan with markers of astrocytic cells and/or neurons and/or detection of the extracellular distribution patterns of neurocan and/or RPTPp/phosphacan is accomplished by RNAscope and/or coimmunostaining.
• the detecting of neurocan and/or RPTPp/phosphacan is accomplished by employing an ELISA assay.
• the colorimetric nature of the ELISA assay provides for a more rapid quantification of the levels of proteins in the sample than is achievable with a Western blot.
• An ELISA assay, or enzyme-linked immunosorbent assay, is a commonly applied biochemical technique known to one of ordinary skill in the art.
• the ELISA assay employed may be a direct ELISA with a single active antibody, an indirect ELISA assay with an unmodified antibody and a secondary reporter antibody, a “sandwich” ELISA with a solid phase-supported antibody and a direct reporter antibody, or any of the other ELISA assays known in the art.
• the method comprises using a multi-dimensional non-linear algorithm to determine if the level (e.g., activity, amount, concentration, concentration of one or more ionized forms, concentration of the neutral form, expression, level, etc.) of at least one molecular form of neurocan and/or RPTPp/phosphacan in the biological sample is statistically different than a comparator.
• level e.g., activity, amount, concentration, concentration of one or more ionized forms, concentration of the neutral form, expression, level, etc.
• the comparator is a level (e g., activity, amount, concentration, concentration of the ionized form, concentration of the neutral form, expression, level, etc.) of at least one molecular form of neurocan and/or RPTPp/phosphacan in a sample obtained from a subject not having an open NTD. In one embodiment, the comparator is a level of at least one molecular form of neurocan and/or RPTPp/phosphacan in a sample obtained from a subject known not to have an open NTD.
• a level e g., activity, amount, concentration, concentration of the ionized form, concentration of the neutral form, expression, level, etc.
• the profile of at least one molecular form of neurocan and/or RPTPp/phosphacan in a subject is compared to a predetermined or comparator profile of at least one molecular form of RPTPp/neurocan and/or phosphacan or reference profile of at least one molecular form of neurocan and/or RPTPp/phosphacan to identify open NTD.
• Control group samples may either be from a normal subject, samples from subjects with a known open NTD, or samples from subjects with no known open NTD.
• the present invention relates to a method for treating open NTD in a subject in need thereof comprising the step of administrating to the subject a treatment for open NTD.
• the fetus upon diagnosis of open NTD, undergoes in utero treatment for correction of the open NTD.
• the in utero treatment includes fetal surgery to repair the open NTD.
• the fetal surgery is an open surgery.
• the fetal surgery is fetoscopic surgery.
• the fetal surgery is a hybrid of open and fetoscopic surgery.
• the in utero treatment includes non-surgical repair.
• the non-surgical repair is transamniotic stem cell therapy.
• treatment includes surgical and stem cell-based therapies.
• the stem cells are derived from placental stem cells.
• the stem cells are placental -derived mesenchymal stem cells. In some embodiments, the stem cells are derived from amniotic fluid. In some embodiments, the stem cells are amniotic fluid- derived mesenchymal stem cells. In some embodiments, the stem cells are umbilical cord- derived. In some embodiments, the stem cells are umbilical cord-derived mesenchymal stem cells. In some embodiments, treatment includes non-stem cells. In some embodiments, the nonstem cells are mesenchymal stromal cells. In some embodiments, the mesenchymal stromal cells are placental-derived. In some embodiments, the mesenchymal stromal cells are umbilical cord- derived. In some embodiments, the mesenchymal stromal cells are placental -derived. In some embodiments, the mesenchymal stromal cells are placental -derived. In some embodiments, the mesenchymal stromal cells are placental -derived.
• the open NTD upon diagnosis of open NTD, is not repaired until after birth. In one embodiment, surgery is used to repair the open NTD after birth. In some embodiments, the surgical repair includes stem cell therapy. In some embodiments, the stem cells are derived from amniotic fluid. In some embodiments, the stem cells are amniotic fluid-derived mesenchymal stem cells. In some embodiments, the stem cells are umbilical cord- derived. In some embodiments, the stem cells are umbilical cord-derived mesenchymal stem cells. In some embodiments, treatment includes non-stem cells. In some embodiments, the nonstem cells are mesenchymal stromal cells. In some embodiments, the mesenchymal stromal cells are placental-derived. In some embodiments, the mesenchymal stromal cells are umbilical cord- derived. In some embodiments, the mesenchymal stromal cells are placental -derived. EXPERIMENTAL EXAMPLES
• CSPGs chondroitin sulfate proteoglycans
• CNS developing central nervous system
• Example 1 Detection of neurocan in amniotic fluid MMC was induced during neurulation by exposure to all-trans RA (Barbe, M. F., et al., 2014, Birth Defects Research A: Clinical & Molecular Teratology, 100:453-462).
• MMC fetuses displayed defects characterized by a pathological opening in the vertebral arch and the overlying skin with the spinal cord exposed at the center of the lesion confined to the lumbosacral area of the fetus (Danzer, E., et al., 2005, Experimental Neurology, 194:467-475; Barbe, M.
• Phosphacan is a secreted spliced extracellular variant of the receptor type protein tyrosine phosphatase beta (RPTPP); also known as protein tyrosine phosphatase receptor type Z1 (PTPRZ1) that is expressed during CNS development and in pathological processes such as tissue injury (Grumet, M., et al., 1996, Perspectives on Developmental Neurobiology, 3:319-330; Maurel, P., et al., 1994, Proceedings of the National Academy of Sciences USA, 91 :2512-2516).
• RPTPP receptor type protein tyrosine phosphatase beta
• PTPRZ1 protein tyrosine phosphatase receptor type Z1
• the analyses aimed to evaluate the presence of neurocan and phosphacan were initially conducted using AF samples pooled from three randomly selected MMC or normal fetuses.
• RA induces MMC in the majority of fetuses with the other littermates appearing normal (Danzer, E., et al., 2005, Experimental Neruology, 194:467-475).
• AF samples from individual E21 fetuses with MMC defects, littermates with no MMC defects after RA exposure, or normal controls were loaded into separate lanes and subjected to western blotting.
• This study identifies the presence of two CNS-associated CSPGs, neurocan and phosphacan in the AF of fetal rats in a clinically relevant model of MMC.
• MMC fetuses showed robust and significantly elevated levels of neurocan and phosphacan in the AF when compared to normal age-matched controls at different developmental time points starting at least as early as E14.
• Normal appearing littermates from RA-exposed cohort showed no increase in the AF levels of neurocan or phosphacan, unlike those with MMC defect.
• Example 3 Aqueous solubility of neurocan and phosphacan
• Example 4 Expression of Neurocan and Phosphacan in MMC spinal cord.
• RNAscope analysis in MMC spinal cord sections using specific probes for the Neurocan and the RPTP fi phosphacan revealed enriched RPTP phosphacan expression in the externally exposed ventricular zone, while the site of neurocan expression was extended to the parenchyma of malformed E14 MMC spinal cords ( Figure 4 A, left panel).
• Tn E21 MMC spinal cords, Neurocan and RPTPp/phosphacan were expressed robustly throughout the spinal cord tissue, with RPTPp/phosphacan expression maintained in the ventricular zone region ( Figure 4 A, right panel).
• RNAscope analysis was combined with coimmunostaining for glutamine synthetase (GS), a marker of astrocytic cells (Anlauf, E., et al., 2013, Front. Endocrinol, 4: 144) and MAP2, a marker of neuronal cells (Dehmelt, L., et al., 2005, Genome Biol, 6:204).
• GS glutamine synthetase
• MAP2 a marker of neuronal cells
• AFP alpha fetoprotein
• AChE acetylcholine esterase
• AFP assessment during the early second trimester is the biochemical marker for open NTDs
• non- neurological fetal disorders are also associated with elevated AFP levels and cannot be excluded based on this marker (Weiss, R. R., et al., 1976, Obstetrics & Gynecology, 47:148-151; Rose, N. & Minneti, M. T., 2009, Global library of women's medicines).
• neurocan and phosphacan are CNS-associated CSPGs robustly expressed during fetal development (Margolis, R. K., et al., 1996, Perspectives on Developmental Neurobiology, 3:273-290; Meyer-Puttlitz, B , et al., 1996, Journal of Comparative Neurology, 366:44-54).
• diagnostic biomarkers specific for neural tissue would allow distinction from non-neurological fetal disorders, therefore increasing the diagnostic accuracy for NTDs.
• AFP tests require specific gestational ages, with the optimal time for AFP testing in the maternal serum between 16-18 weeks of gestation and in the AF between 13-22 weeks of gestation, when serum and AF levels of AFP have been found to be substantially elevated in NTD-affected pregnancies (Palomaki, G. E., et al., 2020, Genetics in Medicine, 22:462-474; Rose N. & Mennuti, M. T., 2009, Global library of women's medicines; Bradley, L. A., et al. 2005 Genetics in Medicine, 7(5):344-54).
• the range of gestational age where AFP levels are markedly elevated is narrow, therefore the interpretation of AFP levels is complicated by adjustment for the fetal age.
• AF levels of neurocan and phosphacan were significantly elevated in comparison to controls from as early as E14 and their content in the AF of MMC fetuses increased with advancing gestational age, while decreased in normal AF.
• differences in the AF levels of neurocan and phosphacan can be used to discriminate between normal fetuses and those affected by the open NTD at all examined gestational ages.
• the prominent differences between MMC and normal fetuses are significantly increased AF levels of neurocan and phosphacan detected in MMC fetuses from as early as E14, with their levels in the AF of MMC fetuses raising rapidly at E16 and reaching the highest levels at E21.
• Antibodies and ELISA assays for human CSPGs are commercially available and currently being studied for quantification of their levels in human bodily fluids (Minta, K. et al., 2019, Clinical Chemistry and Laboratory Medicine, 57: 1565-1573).
• Neurocan and phosphacan are CSPGs expressed in the developing CNS that are secreted locally and assembled in the surrounding extracellular space (Margolis, R.
• neurocan and phosphacan are highly soluble ECM proteins that transfer efficiently from the MMC spinal cord tissue into the AF or detergent-free buffer and are found at exceptionally high levels in the AF of MMC fetuses even at very early gestational ages (E14). This is consistent with previous reports demonstrating low affinity attachment of these CSPGs within the neural ECM and their ease of extraction from the spinal cord tissue using the detergent-free buffer (Deepa, S. S.
• the principal findings indicate that the significant differences in the AF levels of neurocan and phosphacan allow distinction between MMC and normal controls at all examined gestational ages and may constitute potential biomarkers to facilitate early diagnosis of open NTDs. Based on the present results, demonstrating a correlation between the increased AF levels of neurocan and phosphacan and the presence ofMMC defect in fetal rats, further studies of human pregnancies will be required to confirm these associations.
• the identification of an early and specific biomarker can provide information supplementary to diagnostic imaging, beyond what is currently offered by the laboratory tests in the field of early detection and diagnosis of open NTDs.
• CNS-associated proteins such as neurocan and phosphacan in the AF ofMMC fetuses provides new information about changes in the composition of AF in in the presence of the defect and may also contribute to a better understanding of underlying pathophysiology and development of prenatal therapies.
• IU international units
• Lumbar spinal cords were isolated from E21 MMC fetuses collected as described above. Immediately after harvesting, lumbar spinal cord samples from 6-7 randomly selected fetuses were pooled together and collected onto 50 mM Tris-HCl buffer with 0.5% TRITON X- 100 (Fisher Scientific; USA) and protease inhibitor cocktail. Samples were subsequently homogenized, rotated at 4°C for 45 min, and centrifuged for 14.000 x g, 30 min at 4°C. Protein concentration in spinal cord extracts was measured using colorimetric detection and quantification kit PierceTM BCA Protein Assay Kit (Thermo Scientific, USA), and samples were stored at -80°C until further analysis.
• Lumbar spinal cords were isolated from MMC fetuses collected at E14, E16, E18 or E21 as described above. Immediately after harvesting, lumbar spinal cords from 6-7 randomly selected fetuses per age group were pooled together and collected onto 50 mM Tris-HCl buffer with protease inhibitors cocktail. Samples were homogenized, rotated at 4°C for 45 min, and centrifuged for 14.000 x g, 30 min at 4°C (1 st extract).
• the resultant pellets were lysed with buffer composed of 50 mM Tris-HCl with 0.5% TRITON X-100 and protease inhibitors rotated at 4°C for 45 min, and centrifuged for 14.000 x g, 30 min at 4°C (2 nd extract). Protein concentration was measured using colorimetric detection and quantification kit PierceTM BCA Protein Assay Kit, and samples were stored at -80°C until further analysis. Prior to gel electrophoresis, 1 st and 2 nd protein extracts (10 pg each) were digested with ChABC and subjected to western blotting analyses as described below. All analyses were made using spinal cord extracts prepared from at least two independent sets of spinal cord tissue samples per each age group.
• Lumbar spinal cords were isolated from E21 MMC fetuses collected as described above. To determine the solubility of neurocan and phosphacan into the AF, lumbar spinal cords from 6-7 randomly selected fetuses were pooled together and collected into the AF of normal age-matched fetuses. Samples were rotated for 45 min at 4°C and centrifuged at 14,000 x g, 4°C for 30 min. Prior to gel electrophoresis, equal volumes of spinal cord extracts were digested with ChABC and subjected for Western blot analyses as described below. All analyses were made using spinal cord extracts prepared from at least two independent sets of MMC spinal cord tissue samples and AF collected from normal age-matched fetuses.
• Amniotic fluid or spinal cord samples were resolved on 8% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto 0.22 pM nitrocellulose membranes (Li-Cor Biosciences; USA). After transfer, blots were blocked for non-specific binding with 5% Blotting-Grade Blocker (Bio-Rad; USA) in TBS and incubated with one of the following antibodies: 1F6 mouse monoclonal antibody (1 :500; Developmental Studies Hybridoma Bank - DSHB, USA), 650.24 mouse monoclonal antibody (1: 1000; Santa Cruz Biotechnology; USA), and 3F8 mouse monoclonal antibody (1 :250; DSHB, USA).
• 1F6 mouse monoclonal antibody (1 :500; Developmental Studies Hybridoma Bank - DSHB, USA
• 650.24 mouse monoclonal antibody (1: 1000; Santa Cruz Biotechnology; USA
• 3F8 mouse monoclonal antibody (1 :250; DSHB,
• the sizes of the detected proteins were estimated using Precision Plus ProteinTM All Blue Prestained Protein Standards (Bio-Rad; USA). Blots were incubated with goat anti-mouse antibody conjugated to IRDye® 680RD dye (LI-COR Biosciences; USA) and signals were detected using Odyssey CLx Imaging System (LI- COR Biosciences). Image Studio Ver. 3.1 . (LT-COR Biosciences) was used to quantify the signal intensity presented as arbitrary fluorescence units (AFU).
• AFU arbitrary fluorescence units
• Fetal rats were collected as described above and then fixed in 10% neutral buffer formalin (Fisher Scientific; USA) at 4°C. After fixation, samples were equilibrated in 15 % followed by 30% sucrose in PBS at 4°C before embedding in OCT (Sakura Finetek USA, USA), and frozen. Serial 1 pm coronal sections were obtained through the center of the MMC defect and mounted on charged glass slides (Superfrost Plus, Fisher Scientific, USA).
• Sections were then incubated with 1F6 (1 :200), 650.24 (1 :250) or 3F8 (1: 150) antibodies and subsequently with anti-mouse Alexa Fluor® 555 secondary antibody (1 : 1000; Fisher Scientific) and 4’6’-diamidini- 2-phenylindole (DAPI; Millipore Sigma) to visualize cell nuclei. Sections when then mounted with ProLong Gold antifade reagent (Fisher Scientific, USA) and cover slipped. Photographs were taken using Zeiss AxiolO microscope (Zeiss) and Leica SP8 confocal microscope (Leica). All analyses were made using cross sections obtained from the lumbar spinal cord region of three MMC fetuses collected at E21 by examining at least three sections from each fetus.
• RNAscope For RNAscope, sections were processed using an RNAscope® V2 multiplex fluorescent reagent kit (Advanced Cell Diagnostics Inc. ACD, USA) and probes specifically targeting rat Neurocan (Rn-Ncan-C2; ACD, USA) and rat RPTPp/phosphacan (Rn-Ptprzl-C3; ACD, USA, which recognizes all alternatively spliced transcript variants) according to the manufacturer’s protocol. Cell nuclei were stained with DAPI. Sections were then mounted with ProLong Gold antifade reagent and cover-slipped. Photographs were taken using a Leica SP8 confocal microscope.
• RNAscope with coimmunostaining were carried out using an RNA protein codetection ancillary kit (ACD, USA), mouse anti-MAP2 (MAB3418; 1 :100; Millipore, USA), and rabbit antiglutamine synthetase (ab73593; 1 :200; Abeam, USA) antibodies according to the manufacturer’s protocol.
• Cell nuclei were stained with DAPI. Sections when then mounted with ProLong Gold antifade reagent and cover-slipped. Photographs were taken using a Leica SP8 confocal microscope. All analyses were performed using cross sections obtained from the lumbar spinal cord region of three MMC fetuses collected at E21 by examining at least two sections from each fetus.

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