Method for identifying and employing target site blocking oligonucleotides in the treatment of champ1 haploinsufficiency syndrome

Abstract

CHAMP1 haploinsufficiency syndrome is caused by deficiencies in normal CHAMP1 protein expression. Herein are described methods of identifying target site blocking (TSB) oligonucleotides that hybridize to CHAMP1 mRNA and thereby block the binding of inhibitory non-coding RNAs, such as miRNAs. Such TSB oligonucleotides are therefore useful in treating CHAMP1 haploinsufficiency syndrome.

Description

[0001] DESCRIPTION

[0002] METHOD FOR IDENTIFYING AND EMPLOYING TARGET SITE BLOCKING OLIGONUCLEOTIDES IN THE TREATMENT OF CHAMP1 HAPLOINSUFFICIENCY SYNDROME

[0003] PRIORITY CLAIM

[0004] This application claims benefit of priority to U. S. Provisional Application Serial No.

[0005] 63 / 711,257, filed October 24, 2024, the entire contents of which arc hereby incorporated by reference.

[0006] REFERENCE TO A SEQUENCE LISTING

[0007] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on October 21, 2025, is named MESCP0146WO.xml and is 9,990 bytes in size.

[0008] BACKGROUND

[0009] 1. Field of the Disclosure

[0010] The present disclosure relates generally to the fields of medicine, genetics, and molecular biology. More particularly, the disclosure relates to methods of identifying target site blocking (TSB) oligonucleotides and their use in treating CHAMP1 haploinsufficiency syndrome.

[0011] 2. Background

[0012] CHAMP 1 Haploinsufficiency is a genetic condition resulting from mutations or deletions in one copy of the CHAMP1 gene. This condition can lead to a variety of developmental and neurological issues, including intellectual disability, developmental delay, and dysmorphic features. The CHAMP1 protein is important for mitosis and cell division, particularly in the alignment and separation of chromosomes. While its precise roles are still being explored, the root cause of the disorder is the lack of sufficient functional protein to carry out necessary cellular functions. At present, there is a complete lack of interventional therapies to address the root cause of the disease, namely, reduced CHAMP 1 function. SUMMARY

[0013] Thus, in accordance with the present disclosure, there is provided a method of identifying a target site blocking (TSB) oligonucleotide that selectively regulates CHAMP 1 expression comprising (a) designing a candidate oligonucleotide that is complementary to a predicted miRNA binding site in CHAMP1 mRNA; (b) contacting said candidate oligonucleotide with a first cell expressing CHAMP 1; (c) incubating said first cell under conditions supporting protein expression; and (d) comparing the expression of CHAMP1 in said first cell as compared to a control cell expressing CHAMP 1 in the absence of said candidate oligonucleotide, wherein an increase in expression of CHAMP1 in said first cell as compared to said control cell identifies said candidate oligonucleotide as a TSB oligonucleotide. The candidate oligonucleotide may comprise one or more non-natural modifications, such as a backbone modification or a sugar modification.

[0014] The predicted binding site may be a miRNA seed sequence and / or may be in the 5’ UTR or 3’ UTR of said CHAMP1 mRNA. The candidate oligonucleotide may be 15-50 nucleotides in length, 18-50 nucleotides in length, 19-50 nucleotides in length, 20-50 nucleotides in length, 18-30 nucleotides in length, 20-30 nucleotides in length, 19-23 nucleotides in length, 21-23 nucleotides in length, or 19, 21 or 23 nucleotides in length. The first and / or second cell may be a neural progenitor cell (NPC), such as a human NPC (hNPC), such as iPSC-derived NPCs, including patient-derived iPSC-derived NPCs. Step (c) may comprise anti-CHAMPl antibody binding to CHAMP 1. Antibody binding may comprise ELISA or EACS analysis. Step (c) may comprise incubating for 48-72 hours.

[0015] The CHAMP 1 may comprise a detectable marker, such as luciferase or green fluorescent protein. The method may further comprise performing one of more control reactions, such as assessing the effect of said candidate oligonucleotide on non-CHAMPl protein expression, or generating and testing point mutants of said TSB oligonucleotide. The candidate oligonucleotide may be a non-natural / modified oligonucleotide, such as where said candidate oligonucleotide contains one or more non-natural / modified nucleotides that increase stability. The method may further comprise testing an identified TSB oligonucleotide in any in vivo model, such as a mouse model, such as a mouse model for CHAMP 1 Haploinsufficiency syndrome. Two, 3, 4 or more identified TSB oligonucleotides may be tested in the same mouse. The method may further comprise modifying said identified TSB oligonucleotide to increase activity and / or stability and / or to decrease an off-target effect. Also provided is a target site blocking (TSB) oligonucleotide that selectively regulates CHAMP 1 expression identified according to the methods described herein. Further provided is aA target site blocking (TSB) oligonucleotide consisting essentially or consisting of 15-50 nucleotides, that hybridizes to a 5’ or 3’ untranslated region of a CHAMP1 mRNA, and that selectively regulates CHAMP 1 expression identified, wherein said TSB oligonucleotide comprises one or more non-natural / modified nucleotides, such as a backbone modification or sugar modification. The TSB oligonucleotide may be completely complementary over its full length to a portion of the 5’ or 3’ untranslated region of a CHAMP1 mRNA, such as comprising a sequence of as shown in FIG. 4. The TSB oligonucleotide may comprise 1, 2, 3, 4 or 5 mismatches over its full length as compared to a portion of the 5’ or 3’ untranslated region of a CHAMP 1 mRNA but retains complete complementarity to a sequence as shown in FIG. 4. The TSB oligonucleotide may comprise an miRNA seed sequence as shown in FIG. 4, or the sequence of any one of SEQ ID NOS: 2-6.

[0016] In another embodiment, there is provided a pharmaceutic l formulation comprising (a) one or more target site blocking (TSB) oligonucleotides consisting essentially or consisting of about 15-50 nucleotides, that hybridize(s) to a 5’ or 3’ untranslated region of a CHAMP1 mRNA, and that selectively regulates CHAMP1 expression identified, optionally where said TSB oligonucleotide comprises one or more non-natural / modified nucleotides; and (b) a pharmaceutically acceptable buffer, diluent or excipient. The TSB oligonucleotide may be completely complementary over its full length to a portion of the 5’ or 3’ untranslated region of a CHAMP 1 mRNA, such as comprising a sequence of as shown in FIG. 4. The TSB oligonucleotide may comprise 1, 2, 3, 4 or 5 mismatches over its full length as compared to a portion of the 5’ or 3’ untranslated region of a CHAMP1 mRNA but retains complete complementarity to a sequence as shown in FIG. 4. The TSB oligonucleotide may comprise a sequence of as shown in FIG. 4.

[0017] In yet another embodiment there is provided, a method of treating a subject having CHAMP 1 haploinsufficiency syndrome comprising providing a target site blocking (TSB) oligonucleotide of any one of claims 16-19 or a pharmaceutical formulation of claim 20 to said subject. The method may further comprise treating said subject with said TSB oligonucleotide or said pharmaceutical formulation a second time. The TSB oligonucleotide or said pharmaceutical formulation may be administered intracerebroventricularly or intrathecally. The TSB oligonucleotide may be delivered using a modified nanocarrier or exosome capable of binding to and crossing the blood-brain barrier. The TSB oligonucleotide may comprise a sequence of as shown in FIG. 4. The TSB oligonucleotide may be provided by administering an expression construct encoding and expressing the TSB oligonucleotide. The expression construct maybe a viral construct, such as an adeno-associated viral construct.

[0018] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number.

[0019] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0021] FIG. 1. Schematic of the human CHAMP1 3' UTR showing potential microRNA binding sites. Image generated using TargetScan (targetscan.org).

[0022] FIG. 2. microRNAs predicted to regulate human or mouse CHAMP1 expression, predicted using the miRTarBase software (mirtarbase.cuhk.edu.cn).

[0023] FIG. 3. CHAMP 1 3’UTR sequence with targeted binding sites underlined (SEQ ID NO: 1).

[0024] FIG. 4. Table showing the TSB oligonucleotide sequences and modification (SEQ ID NOS: 8, and 2-6; top to bottom), as well as the predicted miRNA binding sites with which they are designed to compete.

[0025] FIG. 5. Western blot showing CHAMP1 protein expression in neural progenitor cells (NPCs) differentiated from human iPSCs. The first two lanes (CHAMP1A and CHAMP1B) represent NPCs derived from patient iPSCs, while the third lane is a CHAMP1 knockout line. The last three lanes (WT-K3, WT-20B, WT-ASE) correspond to wild-type controls. CHAMP 1 (bands about 125 kD) is expressed in all lines except the knockout, detected using an anti-CHAMPl antibody (Sigma Aldrich, HPA008900). The bands between 38 and 50 kD represent beta-actin (Abeam, AB8226), used as a loading control.

[0026] FIG. 6. Details of the cell lines used.

[0027] FIG. 7. Alternative Illustration of CHAMP 1 sequence (SEQ ID NOS: 1 and 7) with predicted binding site (snapgene).

[0028] FIG. 8. Western blot of SK-N-SH cells showing increased CHAMP1 expression with multiple TSBs compared to untransfected (vehicle) and control TSB (at 5uM, collected 96 hours after treatment).

[0029] FIG. 9. Western blot of “CHAMP1A” patient-derived NPCs showing increased CHAMP1 expression with at least 2 TSBs compared to untransfected (vehicle) and control TSB (at 5uM, collected 56 hours after treatment).

[0030] FIG. 10. Western blot of patient-derived “CHAMP 1 A” NPCs showing a dosedependent response to TSB 1. CHAMP 1 protein levels increase progressively with higher TSB concentrations, peaking at 5-10 M, and then decline at 20 p M (treatment for 56 hours). DETAILED DESCRIPTION

[0031] As noted above, CIIAMP1 is at present a largely untreatable disease. The inventor is proposing a promising treatment strategy for CHAMP 1 haploinsufficiency that enhances the natural expression of the CHAMP 1 protein. An approach involves disrupting endogenous regulatory mechanisms that control protein synthesis at the RNA level. MicroRNAs (miRNAs), which are small non-coding RNAs, can attach to messenger RNAs (mRNAs) and generally diminish their protein-producing capability. Targeting this miRNA-mediated silencing could elevate CHAMP 1 protein levels and deliver therapeutic benefit to patients.

[0032] The workflow involves identifying potential miRNAs that regulate a specific messenger RNA by computational prediction, validating these findings in cell lines, and then synthesizing short oligonucleotides that compete with miRNAs for binding to the protein’s mRNA, limiting their silencing ability. These oligonucleotides are called target site blockers (TSBs). For CHAMP1, there is a relatively small number of miRNAs predicted to regulate it (less than 20), which presents an opportunity to expedite the research by testing all possible TSBs simultaneously, potentially speeding up the identification of effective therapeutic interventions. Preliminary analysis in human neural progenitor cells (hNPCs) revealed detectable CHAMP 1 protein levels (FIG. 6), enabling the inventors to assess effects of candidate TSBs to upregulate CHAMP1.

[0033] These and other aspects of the disclosure are described below.

[0034] I. CHAMP1 and CHAMP1 Haploinsufficiency Syndrome

[0035] Chromosome alignment- maintaining phosphoprotein 1 (CHAMP 1), also known as zinc finger protein 828 (ZNF828), is a protein that in humans is encoded by the CHAMP 1 gene. CHAMP1 -associated intellectual disability syndrome, also known as autosomal dominant intellectual disability type 40, is a rare genetic disorder characterized by intellectual disabilities, developmental delays, facial dysmorphisms, and other anomalies caused by mutations in the CHAMP 1 gene in chromosome 13q34. These mutations are most often missense or nonsense mutations. They are usually sporadic, meaning the condition is not inherited from the parents. However, if people with the disorder were to reproduce, they would have a 1 in 2 chance of giving their children a copy of the gene, because the disorder is autosomal dominant, which means that only one copy of a mutated gene (whether inherited or from a spontaneous error in cell division) is needed to pass it on to a child. According to OMIM, only 36 cases have been described in medical literature. Worldwide, there are only around 170 known cases, as confirmed by the CHAMP 1 non-profits CHAMP 1 UK and The CHAMP 1 Research Foundation.

[0036] IL Target Site Blocking (TSB) Oligonucleotides

[0037] In one aspect of the disclosure, there are provided oligonucleotides designed to target and protect a site in the 3’ untranslated region of the CHAMP 1. Exemplary sequences for CHAMP 1 include those in the following accession nos: mRNA - XMJ147430277.1; protein -XP_047286233.1 In a particular embodiment, the target sequence is one as shown in FIG. 4. The TSBs may comprise DNA bases, RNA bases, or non-natural bases. The oligonucleotides will therefore be 10-50, 10-40, 10-30, 10-25, 10-22, 15-50, 15-40, 15-30, 15-25, 15-22, 20-50, 20-40, 20-30, 20-25, 20-22, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length and contain the core sequences, shown in the larger font in FIG. 4. The regions of the TSBs outside these core sequences may contain 1 or more mismatches as compared to the corresponding CHAMP 1 mRNA sequences, such as 1, 2, 3, 4 or 5 mismatches.

[0038] In certain embodiments, oligonucleotides provided herein may comprise one or more modifications to a nucleobase, sugar, and / or intemucleoside linkage, and as such is a modified oligonucleotide. A modified nucleobase, sugar, and / or intemucleoside linkage may be selected over an unmodified form because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets and increased stability in the presence of nucleases. In certain embodiments, the present disclosure provides oligonucleotides comprising linked nucleosides. In such embodiments, nucleosides may be linked together using any intemucleoside linkage.

[0039] The two main classes of intemucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing intemucleoside linkages include, but are not limited to, phosphodiesters (P=O), phosphotriesters, methylphosphonates, methylenephosphonates, vinylphosphonates, phosphonoacetates, thiophosphonoacetates, phosphoramidates, and phosphorothioates (P=S). Representative non-phosphorus containing intemucleoside linking groups include, but are not limited to, amide, triazole, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thionocarbamate (-O-C(O)(NH)-S-): siloxane (-O-Si(H)2-O-); and N, N'-dimethylhydrazine (— CH2— N(CH3)— N(CH3)-). Modified linkages, compared to natural phosphodicstcr linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In certain embodiments, intemucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing intemucleoside linkages are well known to those skilled in the art.

[0040] In certain embodiments, a 2’ -modified nucleoside comprises a 2’ -substituent group selected from F, OCF3, O-CH3 (also referred to as “2’-0Me”), OCH2CH2OCH3 (also referred to as “2’-O-methoxyethyl” or “2’-M0E”), 2'-O(CH2)2SCH3, O-(CH2)2-O- N(CH3)2. -O(CH2)2O(CH2)2N(CH3)2, and O-CH2-C(=O)-N(H)CH3.

[0041] Another non-natural modified oligonucleotide is a locked nucleic acid (LNA), often referred to as inaccessible RNA, The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' and 4' carbons. The bridge “locks” the ribose in the 3’-endo structural conformation, which is often found in the A-form of DN A or RNA. LNA nucleotides can be mixed with DNA or RNA bases in the oligonucleotide whenever desired. Such oligomers are commercially available. The locked ribose conformation enhances base stacking and backbone pre-organization. This significantly increases the thermal stability (melting temperature) of oligonucleotides (Kaur el al., 2006).

[0042] III. Formulation and Administration

[0043] The present disclosure provides pharmaceutical compositions comprising TSB oligonucleotides. Such compositions comprise a prophylactically or therapeutically effective amount of an agent, and a pharmaceutically acceptable earner. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0044] In certain embodiments, the oligonucleotide will be prepared in a suitable diluent, adjusted to pH 7.0-9.0 with acid or base during preparation, and then lyophilized under sterile conditions. The lyophilized modified oligonucleotide is subsequently reconstituted with a suitable diluent, e.g., aqueous solution, such as water or physiologically compatible buffers such as saline solution, Hanks's solution, or Ringer's solution. The reconstituted product is administered as a subcutaneous injection or as an intravenous infusion. The lyophilized drug product may be packaged in a 2 mL Type I, clear glass vial (ammonium sulfate-treated), stoppered with a bromobutyl rubber closure and sealed with an aluminum overseal.

[0045] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, intra-rectal, vaginal, topical or delivered by mechanical ventilation.

[0046] Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0047] The TSB oligonucleotide may also be delivered via expression from an expression vector. The vector may be a non-viral vector, such as a plasmid, or a viral vector, such as a retroviral vector, a lentiviral vector, a pox viral vector, a herpesviral vector, an adenoviral vector or an adeno-associated viral (AAV) vector. The AAV vector may be replication-defective or conditionally replication defective and / or may be a recombinant AAV vector. The AAV vector may comprise a sequence isolated or derived from an AAV vector of serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6),7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV10), 11 (AAV11)

[0048] Generally, ingredients of the compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0049] IV. Assays and Kits

[0050] A. Assays

[0051] In further embodiments, the present disclosure concerns immunodetection methods for detecting and / or quantifying CHAMP 1. A wide variety of assay formats are contemplated, but specifically those that would be used to detect CHAMP 1. Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. In particular, a competitive assay for the detection and quantitation of CHAMP 1 also is provided. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). In general, the immunobinding methods include obtaining a sample containing CHAMP 1 and contacting the sample with a first antibody in under conditions effective to allow the formation of immunocomplexes.

[0052] The immunobinding methods also include methods for detecting and quantifying the amount of CHAMP 1 or related components in a sample and the detection and quantification of any immune complexes formed during the binding process. Here, one would obtain a sample suspected of containing CHAMP 1 and contact the sample with an antibody that binds CHAMP 1 or components thereof, followed by detecting and quantifying the amount of immune complexes formed under specific conditions. Contacting the chosen biological sample with the antibody under effective conditions and for a period sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to CHAMP1 present. After this time, the sample-antibody composition, such as an ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected. In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. Patents concerning the use of such labels include U. S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Of course, one may find additional advantages using a secondary binding ligand such as a second antibody and / or a biotin / avidin ligand binding arrangement, as is known in the art.

[0053] The antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a “secondary” antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

[0054] Further methods include the detection of primary immune complexes by a two-step approach. A second binding ligand, such as an antibody that has binding affinity for the antibody, is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide signal amplification if this is desired.

[0055] One method of immunodetection uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and a second antibody is then used to detect the biotin attached to the complexed biotin. In that method, the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody / antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin. This second step antibody is labeled, for example, with an enzyme that can be used to detect the presence of the antibody / antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible.

[0056] Another known method of immunodetection takes advantage of the immuno-PCR (Polymerase Chain Reaction) methodology. The PCR method is like the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA / biotin / streptavidin / antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.

[0057] B. Kits

[0058] In still further embodiments, the present disclosure concerns kits for use with the methods described herein. TSB oligonucleotides may be included in the kit. Kits for immunodetection are also contemplated and will thus comprise, in suitable container means, one or more CHAMP 1 binding agent.

[0059] The kits may further comprise a suitably aliquoted composition of the TSB oligonucleotide(s) and or CHAMP1 binding agents, whether in dry (e.g., lyophilized) or in aqueous form.

[0060] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the TSB oligonucleotide(s) may be placed, or preferably, suitably aliquoted. Such containers / devices for mixing, diluting and administering the TSB oligonucleotides / CHAMPl binding agents. Directions for mixing, diluting and administering the TSB oligonucleotides may also be included.

[0061] The kits of the present disclosure will also typically include a means for containing the TSB oligonucleotide(s) / CHAMPl binding agents and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

[0062] VI. References

[0063] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0064] Kaur et al., Biochemistry, 45:7347-55, 2006.

Claims

WHAT IS CLAIMED:

1. A method of identifying a target site blocking (TSB) oligonucleotide that selectively regulates CHAMP 1 expression comprising:(a) designing a candidate oligonucleotide that is complementary to a predicted miRNA binding site in CHAMP1 mRNA;(b) contacting said candidate oligonucleotide with a first cell expressing CHAMP 1; (c) incubating said first cell under conditions supporting protein expression; and (d) comparing the expression of CHAMP 1 in said first cell as compared to a control cell expressing CHAMP 1 in the absence of said candidate oligonucleotide, wherein an increase in expression of CHAMP1 in said first cell as compared to said control cell identifies said candidate oligonucleotide as a TSB oligonucleotide.

2. The method of claim 1, wherein said predicted binding site is a miRNA seed sequence and / or is in the 5’ UTR or 3’ UTR of said CHAMP1 mRNA.

3. The method of claim 1 or claim 2, wherein said candidate oligonucleotide is 15-50 nucleotides in length, 18-50 nucleotides in length, 19-50 nucleotides in length, 20-50 nucleotides in length, 18-30 nucleotides in length, 20-30 nucleotides in length, 19-23 nucleotides in length, 21-23 nucleotides in length, or 19, 21 or 23 nucleotides in length.

4. The method of any one of claims 1-3, wherein said first and / or second cell is a neural progenitor cell (NPC), such as a human NPC (hNPC), such as iPSC-derived NPCs, including patient-derived iPSC-derived NPCs.

5. The method of any one of claims 1-4, wherein step (c) comprises anti-CHAMPl antibody binding to CHAMP 1.

6. The method of claim 5, wherein antibody binding comprises ELISA or FACS analysis.

7. The method of any one of claims 1-6, wherein step (c) comprises incubating for 48-72 hours.

8. The method of any one of claims 1-8, wherein the CHAMP 1 comprises a detectable marker.

9. The method of claim 8, wherein the detectable marker is luciferase or green fluorescent protein.

10. The method of any one of claims 1-9, further comprising performing one of more control reactions, such as assessing the effect of said candidate oligonucleotide on non- CHAMP1 protein expression, or generating and testing point mutants of said TSB oligonucleotide.

11. The method of any one of claims 1-10, wherein said candidate oligonucleotide is a non- natural / modified oligonucleotide, such as where said candidate oligonucleotide contains one or more non-natural / modified nucleotides that increase stability.

12. The method of any one of claims 1-11, further comprising testing an identified TSB oligonucleotide in any in vivo model, such as a mouse model, such as a mouse model for CHAMP 1 Haploinsufficiency syndrome.

13. The method of claim 12, wherein 2, 3, 4 or more identified TSB oligonucleotides are tested in the same mouse.

14. The method of any one of claims 1-13, further comprising modifying said identified TSB oligonucleotide to increase activity and / or stability and / or to decrease an off-target effect.

15. The method of claims 1-14, wherein the candidate oligonucleotide comprises one or more non-natural modifications, such as a backbone modification or a sugar modification.

16. A target site blocking (TSB) oligonucleotide that selectively regulates CHAMP1 expression identified according to the method of any one of claims 1-15.

17. A target site blocking (TSB) oligonucleotide consisting essentially or consisting of 15- 50 nucleotides, that hybridizes to a 5’ or 3’ untranslated region of a CHAMP 1 mRNA, and that selectively regulates CHAMP 1 expression identified, wherein said TSB oligonucleotide comprises one or more non-natural / modified nucleotides, such as a backbone modification or sugar modification.

18. The TSB oligonucleotide of claim 17, wherein said TSB oligonucleotide is completely complementary over its full length to a portion of the 5’ or 3’ untranslated region of a CHAMP 1 mRNA, such as comprising a sequence of as shown in FIG. 4.

19. The TSB oligonucleotide of claim 17, wherein said TSB oligonucleotide comprises 1, 2, 3, 4 or 5 mismatches over its full length as compared to a portion of the 5’ or 3’ untranslated region of a CHAMP 1 mRNA but retains complete complementarity to a sequence as shown in FIG. 4.

20. The TSB oligonucleotide of claim 17, wherein the TSB oligonucleotide comprises an miRNA seed sequence as shown in FIG. 4, or the sequence of any one of SEQ ID NOS: 2-6.

21. A pharmaceutical formulation comprising:one or more target site blocking (TSB) oligonucleotides according to any one of claims 16-20; and a pharmaceutically acceptable buffer, diluent or excipient.

22. A method of treating a subject having CHAMP 1 haploinsufficiency syndrome comprising providing a target site blocking (TSB) oligonucleotide of any one of claims 16-19 or a pharmaceutical formulation of claim 20 to said subject.

23. The method of claim 22, further comprising treating said subject with said TSB oligonucleotide or said pharmaceutical formulation a second time.

24. The method of claim 22 or claim 23, wherein said TSB oligonucleotide or said pharmaceutical formulation is administered intracerebroventricularly or intrathecally.

25. The method of any one of claims 22-24, wherein said TSB oligonucleotide is delivered using a modified nanocarrier or exosome capable of binding to and crossing the bloodbrain barrier.

26. The method of any one of claims 22-25, wherein the TSB oligonucleotide is provided by administering an expression construct encoding and expressing the TSB oligonucleotide.

27. The method of claim 26, wherein the expression construct is a viral construct.

28. The method of claim 27, wherein the viral construct is an adeno-associated viral construct.