Methods of producing CDKL5 proteins and uses of the same

Abstract

The present disclosure provides novel methods of producing CDKL5 proteins, novel CDKL5 antibodies, and uses of the same including for in vitro detection assays.

Description

METHODS OF PRODUCING CDKL5 PROTEINS AND USES OF THE SAME CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 535,743, filed on August 31, 2023, the entire disclosure of which is incorporated by reference herein in its entirety for all purposes. SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on August 30, 2024, is named ULTA-029WO_SL.xml and is 131,100 bytes in size. TECHNICAL FIELD OF THE INVENTION

[0003] The present disclosure relates generally to methods of producing soluble and full-length recombinant human cyclin-dependent kinase-like 5 (CDKL5) proteins and methods of purifying and processing the same. BACKGROUND

[0004] CDKL5 deficiency disorder (CDD) is a rare neurodevelopmental disease caused by mutations in the CDKL5 gene which can manifest in a broad range of clinical symptoms and severity. Hallmarks of CDD include infantile-onset refractory epilepsy, developmental delay, intellectual disability, visual impairment, lack of speech, hypotonia, motor dysfunction, sleep disturbances, gastrointestinal dysfunction, and breathing difficulties. Although rare, the occurrence is believed to be approximately 1 in 40,000 - 60,000 live births, making it one of the most common forms of genetic epilepsy.

[0005] CDD is caused by pathogenic variants in the CDKL5 gene that include deletions, truncations, splice variants, and missense mutations. See Olson et al., 2019, Pediatric Neurology 97: 18-25. These variants can reduce the amount of functional CDKL5 protein and / or diminish its activity in neurons. More than 150 mutations in the CDKL5 gene have been found to cause CDD. See Del Rosso et al., 2017, The EuroBiotech Journal 1(2): 122-127. The CDKL5 gene encodes a cyclin-dependent kinase-like 5 (CDKL5) protein that is essential for normal brain development and function. The CDKL5 protein is involved in the formation, growth, and migration of neurons in the brain. It is widely expressed in the brain, predominantly in neurons and dendrites, with roles in cell proliferation, neuronal migration, axonal outgrowth, dendritic morphogenesis, and synapse development.

[0006] To date, there is no treatment that addresses the underlying cause of CDD, namely the deficiency of CDKL5. Accordingly, a therapeutic approach that addresses the underlying cause of the disease, the deficiency of functional CDKL5, is urgently needed. Delivery of human CDKL5 in patients by administration of human CDKL5 encoding recombinant adeno- associated viral vectors (rAAV-CDKL5) or in the form of pure full-length CDKL5, are therapeutic options being investigated. Additionally, full-length CDKL5 in native conformation is necessary to raise detection antibodies in other species like mice and rabbits, which can then be used to evaluate efficacy of therapeutic interventions for expressing human CDKL5 in humans and non-human animal studies. At present, there are no known examples of successfully making and purifying the active full-length protein. The expression of a full- length human CDKL5 protein in a host cell is challenging due to the presence of intrinsically disordered domains in the CDKL5 protein, such that when produced recombinantly, CDKL5 is either subject to a massive proteolytic attack by host-encoded proteases or tends to from aggregates.

[0007] The present application provides methods to express, purify and process CDKL5 proteins from a mammalian system. SUMMARY

[0008] The present disclosure provides reliable methods for producing soluble, non-denatured recombinant human CDKL5 (rhCDKL5) proteins, including truncated and engineered versions of human CDKL5. The methods may involve, for example, expressing and / or purifying the soluble, non-denatured rhCDKL5 protein. Also provided are methods of using said rhCDKL5 proteins in in vitro, diagnostic, and / or analytical assays.

[0009] These methods and the properties of the provided rhCDKL5 enable the characterization and use of these rhCDKL5 in vitro, including in potency assays, immunogenicity assays, and other clinical and diagnostic tool development necessary for evaluation and therapeutic advancement of clinical and pre-clinical engineered human CDKL5 proteins.

[0010] The present disclosure provides a method of producing a recombinant human cyclin- dependent kinase-like 5 (rhCDKL5) protein in solution, the rhCDKL5 protein comprising a CDKL5 sequence comprising an N-terminal catalytic domain and a C-terminal regulatory domain, and at least one intracellular expression tag, wherein the C-terminal regulatory domain comprises a nuclear localization signal 1 domain and a nuclear localization signal 2 domain,the method comprising the steps of: (a) transfecting a host cell culture with a nucleic acid encoding the rhCDKL5 protein; (b) expressing the rhCDKL5 protein in the host cell culture; (c) generating a whole cell lysate by incubating the host cell culture with a lysis buffer; (d) separating soluble fraction comprising the rhCDKL5 protein and insoluble fraction from the whole cell lysate; and (e) isolating the rhCDKL5 protein from the soluble fraction.

[0011] In some embodiments, the CDKL5 sequence of the intracellular expression tagged rhCDKL5 protein comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the CDKL5 sequence of the intracellular expression tagged rhCDKL5 protein comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the CDKL5 sequence of the intracellular expression tagged rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 1. In some embodiments, the CDKL5 sequence of the intracellular expression tagged rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 2.

[0012] In some embodiments, the rhCDKL5 protein comprises at least one intracellular expression tag linked to the N-terminus and / or the C-terminus of the rhCDKL5 protein. In some embodiments, the rhCDKL5 protein comprises at least one intracellular expression tag linked to the N-terminus of the rhCDKL5 protein. In some embodiments, the rhCDKL5 protein comprises at least one intracellular expression tag linked to the C-terminus of the rhCDKL5 protein. In some embodiments, the at least one intracellular expression tag is selected from: (a) a green fluorescent protein (GFP) tag; (b) a maltose binding protein (MBP) tag; and (c) a combination thereof. In some embodiments, the rhCDKL5 protein comprises a GFP tag or an MBP tag linked to the N-terminus of the rhCDKL5 protein. In some embodiments, the rhCDKL5 protein comprises a GFP tag linked to the N-terminus of the rhCDKL5 protein. In some embodiments, the rhCDKL5 protein comprises an MBP tag linked to the N-terminus of the rhCDKL5 protein. In some embodiments, the rhCDKL5 protein comprises a GFP tag or an MBP tag linked to the C-terminus of the rhCDKL5 protein. In some embodiments, the rhCDKL5 protein comprises an MBP tag linked to the C-terminus of the rhCDKL5 protein. Insome embodiments, the rhCDKL5 protein comprises a GFP tag linked to the C-terminus of the rhCDKL5 protein.

[0013] In some embodiments, the rhCDKL5 protein comprises a GFP tag linked to the N- terminus and an MBP tag linked to the C-terminus of the rhCDKL5 protein. In some embodiments, the rhCDKL5 protein comprises an MBP tag linked to the N-terminus and a GFP tag linked to the C-terminus of the rhCDKL5 protein. In some embodiments, the rhCDKL5 protein comprises an MBP tag linked to the N-terminus and a GFP tag linked to the C-terminus of rhCDKL5 protein. In some embodiments, the rhCDKL5 protein comprises a GFP tag linked to the N-terminus and an MBP tag linked to the C-terminus of the rhCDKL5 protein.

[0014] In some embodiments of the methods of the present disclosure, at least one intracellular expression tag is linked to the rhCDKL5 protein by a protease cleavage site linker. In some embodiments, the protease cleavage site linker is a tobacco etch virus (TEV) protease cleavage site linker. In some embodiments, the intracellular expression tag is linked to the N-terminus of the rhCDKL5 protein by a TEV protease cleavage site linker. In some embodiments, the intracellular expression tag is linked to the C-terminus of the rhCDKL5 protein by a TEV protease cleavage site linker. In some embodiments, the intracellular expression tag is linked to the N-terminus and the C-terminus of the rhCDKL5 protein by a TEV protease cleavage site linker.

[0015] In some embodiments, an MBP tag is linked to the N-terminus of the rhCDKL5 protein by a TEV protease cleavage site linker and a GFP tag is linked to the C-terminus of the rhCDKL5 protein by a TEV protease cleavage site linker. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 4. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 4.

[0016] In some embodiments, a GFP tag is linked to the N-terminus of the rhCDKL5 protein by a TEV protease cleavage site linker and an MBP tag is linked to the C-terminus of the rhCDKL5 protein by a TEV protease cleavage site linker. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to an amino acid sequence according to SEQ ID NO: 5. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 5.

[0017] In some embodiments, expressing the rhCDKL5 protein in the host cell culture comprises maintaining the host cell culture under conditions adequate to express the rhCDKL5 protein for about 48 hours to about 72 hours (e.g., about 48 hours to about 52 hours, about 52 hours to about 56 hours, about 56 hours to about 60 hours, about 60 hours to about 64 hours, about 64 hours to about 68 hours, about 68 hours to about 72 hours, about 52 hours to about 68 hours, about 56 hours to 64 hours, and all integers including and between 48 hours to 72 hours).

[0018] In some embodiments, maintaining the host cell culture under conditions adequate to express the rhCDKL5 protein comprises maintaining the host cell culture at about 32oC to about 37oC (about 32oC to about 33oC, about 33oC to about 34oC, about 34oC to about 35oC, about 35oC to about 36oC, about 36oC to about 37oC, about 32oC to about 34oC, about 33oC to about 36oC, about 34oC to about 37oC, and all integers including and between 32oC and 37oC).

[0019] In some embodiments, the step of expressing the rhCDKL5 protein in the host cell culture comprises maintaining the host cell culture under conditions adequate to express the rhCDKL5 protein in a cell culture medium comprising at least one expression enhancer. In some embodiments, the at least one expression enhancer comprises aromatic carboxylic acids, an aliphatic carboxylic acid, a hydroxamic valproic acid, hydroxamic acids, a hydroxamic derivative of valproic acid, an acetamide, sodium butyrate, or a derivative of any one of the foregoing. In some embodiments, the at least one expression enhancer is sodium butyrate or valproic acid. In some embodiments, the at least one expression enhancer is sodium butyrate. In some embodiments, the at least one expression enhancer is valproic acid.

[0020] In some embodiments, the lysis buffer is any one of Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), octylphenoxypolyethoxyethanol (Nonidet P-40), and ammonium-chloride-potassium (ACK) lysis buffer. In some embodiments, the lysis buffer comprises a chelating agent. In some embodiments, the lysis buffer comprises a chelating agent that is any one of ethylenediamine- tetraacetic acid disodium salt (EDTA), ethylene glycol-O, O’-bis(2-aminoethyl)-N, N, N’, N’- tetraacetic acid (EGTA), N-(2-Hydroxyethyl)ethylenediamine-N, N’, N’-triacetic acid trisodium salt (HEDTA), nitrilotriacetic acid (NTA), and Potassium Citrate.

[0021] In some embodiments, prior to incubating the host cell culture with the lysis buffer, the host cell culture is maintained for an amount of time such that the percentage of live cells (viability) of the host cell culture post-transfection with a nucleic acid encoding the rhCDKL5 protein is any one of: (a) at least 50% (e.g., at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%); (b) at least 60% (e.g., at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%); (c) at least 70% (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%); (d) at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%); (e) at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%); or (f) at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%). The viability of the host cell culture may be determined by a microscopy-based assay, colorimetry-based assay, flow-cytometry-based assay, or a combination thereof.

[0022] In some embodiments, prior to incubating the host cell culture with the lysis buffer, the host cell culture is maintained for an amount of time such that the percentage of transfected cells in the host cell culture post-transfection with a nucleic acid encoding the rhCDKL5 protein is any one of: (a) at least 50% (e.g., at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%); (b) at least 60% (e.g., at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%); (c) at least 70% (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%); (d) at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%); (e) at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%); or (f) at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%). The percentage of transfected cells may be determined by a fluorescence microscopy-based assay, immunostaining-based assay, flow-cytometry-based assay, or a combination thereof.

[0023] In some embodiments, separating insoluble and soluble fractions from the whole cell lysate comprises centrifuging of the cell lysate at about 1100×g to about 12000×g (e.g., 1100xg to 2000xg, 2000xg to 3000xg, 3000xg to 4000xg, 4000xg to 5000xg, 5000xg to 6000xg, 6000xg to 7000xg, 7000xg to 8000xg, 8000xg to 9000xg, 9000xg to 10000xg, 10000xg to 11000xg, 11000xg to 12000xg, 2000xg to 11000xg, 3000xg to 10000xg, 4000xg to 9000xg, 5000xg to 8000xg, 6000xg to 8000xg, and all integers between and including 1100xg and12000xg) for about 5 min to about 1 hour (e.g., 5 min to 10 min, 10 min to 20 min, 20 min to 30 min, 30 min to 50 min, 50 min to 1 hour, 10 min to 50 min, 20 min to 40 min, 30 min to 50 min, and all integers between and including 5 min and 1 hour).

[0024] In some embodiments, isolating the rhCDKL5 protein from the soluble fraction comprises using an affinity-based chromatography resin. In some embodiments, isolating the rhCDKL5 protein from the soluble fraction comprises using an anti-MBP protein tag-based chromatography resin. In some embodiments, isolating the rhCDKL5 protein from the soluble fraction comprises using an anti-CDKL5 protein tag-based chromatography resin. In some embodiments, the affinity-based chromatography resin is a dextrin crosslinked to agarose affinity resin.

[0025] Also provided herein are methods for producing the rhCDKL5 protein linked to at least one intracellular expression tag, wherein the rhCDKL5 protein comprises a biotinylable peptide tag at the N-terminus or the C-terminus of the rhCDKL5 protein. In some embodiments, the rhCDKL5 protein comprises a biotinylable peptide tag at the N-terminus of the rhCDKL5 protein. In some embodiments, the rhCDKL5 protein comprises a biotinylable peptide tag at the C-terminus of the rhCDKL5 protein. In some embodiments, the biotinylable peptide tag comprises an amino acid sequence according to SEQ ID NO: 14. In some embodiments, the biotinylable peptide tag according to SEQ ID NO: 14 is located between an intracellular expression tag and the C-terminus of the rhCDKL5 protein. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence according to any one of SEQ ID NOs: 15-18. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 15. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 16. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 17. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an aminoacid sequence according to SEQ ID NO: 18. In some embodiments, the biotinylable peptide tag is located between an intracellular expression tag and the C-terminus of the rhCDKL5 protein. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 18.

[0026] In some embodiments, the method further comprises linking the biotinylable peptide tag of the rhCDKL5 protein to a biotin compound or biotin. In some embodiments, the method further comprises using an intracellular biotin ligase for linking the biotinylable peptide tag of the rhCDKL5 protein to a biotin compound or biotin. In some embodiments, the cells in the host cell culture express E. coli biotin ligase. In some embodiments, the E. coli biotin ligase is BirA.

[0027] Also provided herein are methods for producing a rhCDKL5 protein linked to at least one intracellular expression tag, wherein the rhCDKL5 protein comprises a partner 1 peptide tag comprising an amino acid sequence according to any one of SEQ ID NOs: 30-32 or a fragment thereof that can bind irreversibly to a partner 2 protein comprising an amino acid sequence according to any one of SEQ ID NOs: 33-35 or a fragment thereof, that is labeled with a detectable moiety.

[0028] In some embodiments, the partner 1 peptide tag or a fragment thereof is linked to the N-terminus of a rhCDKL5 protein comprising an amino acid sequence according to SEQ ID NO: 5. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 5. In some embodiments, the rhCDKL5 protein linked to a partner 1 peptide tag or fragment thereof comprises an amino acid sequence according to SEQ ID NO: 22. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 22. In some embodiments, the partner 1 peptide tag or a fragment thereof is located between the GFP-TEV linker and the N-terminus of the CDKL5 sequence in the rhCDKL5 protein comprising an amino acid sequence according to SEQ ID NO: 5. In some embodiments, the rhCDKL5 protein linked to a partner 1 peptide tag or fragment thereof comprises an amino acid sequence according to SEQ ID NO: 23. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 23. In some embodiments, the partner 1 peptide tag or a fragment thereof is linked to the C-terminus of a rhCDKL5 protein comprising an amino acid sequence according to SEQ ID NO: 5. In some embodiments, the rhCDKL5 protein linked to a partner 1 peptide tag or fragment thereof comprises an amino acid sequence according to SEQ ID NO: 24. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 24. In some embodiments, the partner 1 peptide tag or a fragment thereof is located between the C-terminus of the CDKL5 sequence and the MBP- TEV linker in the rhCDKL5 protein comprising an amino acid sequence according to SEQ ID NO: 5. In some embodiments, the rhCDKL5 protein linked to a partner 1 peptide tag or fragment thereof comprises an amino acid sequence according to SEQ ID NO: 25. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 25.

[0029] In some embodiments, the rhCDKL5 protein linked to at least one intracellular expression tags further comprises a step of contacting the rhCDKL5 protein comprising a partner 1 peptide tag with a partner 2 protein labelled with Ruthenium (Ru). In some embodiments, the partner 2 protein comprises an amino acid sequence according to any one of SEQ ID Nos: 33-35.

[0030] The present disclosure also provides a method of producing a recombinant human cyclin-dependent kinase-like 5 (rhCDKL5) protein in solution, the rhCDKL5 protein comprising: a CDKL5 sequence comprising an N-terminal catalytic domain and a C-terminal regulatory domain, and at least one secretion signal tag; wherein the C-terminal regulatory domain comprises a nuclear localization signal 1 domain and a nuclear localization signal 2 domain, the method comprising: (a) transfecting a host cell culture with a nucleic acid encoding the rhCDKL5 protein; (b) expressing the rhCDKL5 protein in the host cell culture; and (c) separating a supernatant from the host cell culture, wherein the supernatant comprises the expressed rhCDKL5 protein.

[0031] In some embodiments, the CDKL5 sequence of the at least one secretion signal tag linked rhCDKL5 protein comprises an amino acid sequence having at least 90% (e.g., at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the CDKL5 sequence of the at least one secretion signal tag linked rhCDKL5 protein comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the CDKL5 sequence of the one or more secretion tag linked rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 1. In some embodiments, the CDKL5 sequence of the one or more secretion tag linked rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 2.

[0032] In some embodiments, the at least one secretion signal tag is linked to the N-terminus and / or the C-terminus of the rhCDKL5 protein. In some embodiments, the secretion signal tag is selected from: a kappa light chain signal sequence tag, a heavy chain signal sequence tag, and a hemagglutinin signal sequence tag. In some embodiments, the kappa light chain signal sequence tag comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence of SEQ ID NO: 6. In some embodiments, the kappa light chain signal sequence tag comprises an amino acid sequence according to SEQ ID NO: 6. In some embodiments, the heavy chain signal sequence tag comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence of SEQ ID NO: 7. In some embodiments, the heavy chain signal sequence tag comprises an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the hemagglutinin signal sequence tag comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence of SEQ ID NO: 8. In some embodiments, the hemagglutinin signal sequence tag comprises an amino acid sequence according to SEQ ID NO: 8.

[0033] In some embodiments, the secretion signal tag linked rhCDKL5 protein comprises an affinity purification tag. In some embodiments, the affinity purification tag is linked to the N- terminus of the rhCDKL5 protein when the rhCDKL5 protein has the at least one secretionsignal tag linked to the C-terminus of the rhCDKL5 protein. In some embodiments, the affinity purification tag is linked to the C-terminus of the rhCDKL5 protein when the rhCDKL5 protein has the at least one secretion signal tag linked to the N-terminus of the rhCDKL5 protein.

[0034] In some embodiments, the affinity purification tag is selected from: a polyhistidine tag, a polyarginine tag, a glutathione-S-transferase tag, a DYKDDDDK (SEQ ID NO: 29) peptide tag, a streptavidin-binding peptide tag, a peptide tag comprising an amino acid sequence according to any one of SEQ ID NOs: 36 or 37, a calmodulin binding peptide tag, a maltose- binding tag, and a cellulose-binding tag. In some embodiments, the affinity purification tag is a polyhistidine tag. In some embodiments, the polyhistidine tag is a 6xHIS tag (SEQ ID NO: 21).

[0035] In some embodiments, the method comprises purifying the rhCDKL5 protein from the supernatant using: (a) a nickel-nitrilotriacetic acid Ni-NTA chromatography resin; (b) a cross- linked dextran chromatography resin; (c) an immobilized glutathione resin; (d) an anti- DYKDDDDK (SEQ ID NO: 29) peptide tag antibody resin; (e) a streptavidin resin; (f) a biotin resin; (g) an immobilized chitin resin; (h) a cellulose resin; (i) a maltose resin; or (j) a calmodulin resin. In some embodiments, the method comprises purifying the rhCDKL5 protein from the supernatant using a Ni-NTA chromatography resin.

[0036] In some embodiments, the rhCDKL5 protein comprises: (a) a kappa light chain signal sequence tag linked to the N-terminus of the rhCDKL5 protein and a 6xHIS tag (SEQ ID NO: 21) linked to the C-terminus of the rhCDKL5 protein; (b) a heavy chain signal sequence tag linked to the N-terminus of the rhCDKL5 protein and a 6xHIS tag (SEQ ID NO: 21) linked to the C-terminus of the rhCDKL5 protein; or (c) a hemagglutinin signal sequence tag linked to the N-terminus of the rhCDKL5 protein and a 6xHIS tag (SEQ ID NO: 21) linked to the C- terminus of the rhCDKL5 protein. In some embodiments, the kappa light chain signal sequence tag comprises an amino acid sequence according to SEQ ID NO: 6. In some embodiments, the heavy chain signal sequence tag comprises an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the hemagglutinin signal sequence tag comprises an amino acid sequence according to SEQ ID NO: 8. In some embodiments, the rhCDKL5 protein comprises a kappa light chain signal sequence tag linked to the N-terminus and a 6xHIS tag (SEQ ID NO: 21) linked to the C-terminus of the rhCDKL5 protein. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to an amino acid sequence according to SEQ ID NO: 9. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 9. In some embodiments, the rhCDKL5 protein comprises a heavy chain signal sequence tag linked to the N-terminus and a 6xHIS tag (SEQ ID NO: 21) linked to the C-terminus of the rhCDKL5 protein. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 10. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 10. In some embodiments, the rhCDKL5 protein comprises a hemagglutinin signal sequence tag linked to the N-terminus and a 6xHIS tag (SEQ ID NO: 21) linked to the C-terminus of the rhCDKL5 protein. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 11. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 11.

[0037] In some embodiments, the method further comprises expressing the rhCDKL5 protein in the host cell culture comprising maintaining the host cell culture under conditions adequate to express the rhCDKL5 protein for a duration up to 7 days (e.g., up to 1 day, up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days, and up to 7 days). In some embodiments, the method comprises the step of isolating or separating the supernatant comprising the expressed rhCDKL5 protein from the host cell culture is repeated or continuous throughout the duration of expressing the rhCDKL5 protein in the host cell culture. In some embodiments, the method comprises the step of isolating or separating the supernatant comprising the rhCDKL5 protein from the host cell culture every 1 day for up to 7 days. In some embodiments, the method comprises isolating or separating the supernatant comprising the rhCDKL5 protein from the host cell culture on the 2nd, 4th, and / or 6thday. In some embodiments, the method comprises isolating or separating the supernatant containing the rhCDKL5 protein from the host cell culture on the 1st, 3rd, 5thand / or 7thday.

[0038] In some embodiments, the method comprises maintaining the host cell culture under conditions adequate to express the rhCDKL5 protein. In some embodiments, maintaining the host cell culture under conditions adequate to express the rhCDKL5 protein comprises maintaining the culture at about 32oC to about 37oC (about 32oC to about 33oC, about 33oC toabout 34oC, about 34oC to about 35oC, about 35oC to about 36oC, about 36oC to about 37oC, about 32oC to about 34oC, about 33oC to about 36oC, about 34oC to about 37oC, and all integers including and between 32oC and 37oC). In some embodiments, the host cell culture is maintained at about 37oC.

[0039] The present disclosure also provides a method of producing a recombinant human cyclin-dependent kinase-like 5 (rhCDKL5) protein in solution, the rhCDKL5 protein comprising: a CDKL5 sequence comprising an N-terminal catalytic domain and a C-terminal regulatory domain, and an exosome trafficking tag; wherein the C-terminal regulatory domain comprises a nuclear localization signal 1 domain and a nuclear localization signal 2 domain, the method comprising the steps of: (a) transfecting a host cell culture with a nucleic acid encoding the rhCDKL5 protein; (b) expressing the rhCDKL5 protein in the host cell culture; and (c) separating a supernatant from the host cell culture, wherein the supernatant comprises exosomes comprising the expressed rhCDKL5 protein.

[0040] In some embodiments, the exosome trafficking tag linked rhCDKL5 protein comprises a CDKL5 sequence comprising an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the exosome trafficking tag linked rhCDKL5 protein comprises a CDKL5 sequence comprising an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the CDKL5 sequence comprises an amino acid sequence according to SEQ ID NO: 1. In some embodiments, the CDKL5 sequence comprises an amino acid sequence according to SEQ ID NO: 2.

[0041] In some embodiments, the exosome trafficking tag is an exosome-targeting viral peptide or a fragment thereof. In some embodiments, the exosome trafficking tag is: (a) an HIV GAG protein or a fragment thereof; (b) an HIV NEF peptide; (c) an HTLV-1 transactivator protein (Tax); (d) an HCV core protein; or (e) an HCV glycoprotein E2. In some embodiments, the exosome trafficking tag is linked to the N-terminus or the C-terminus of the rhCDKL5 protein.

[0042] In some embodiments, the exosome-targeting viral peptide is an HIV GAG protein or a fragment thereof. In some embodiments, the HIV GAG protein or a fragment thereof is linked to the N-terminus of the rhCDKL5 protein. In some embodiments, the HIV GAG protein or a fragment thereof is linked to the C-terminus of the rhCDKL5 protein. In some embodiments, the HIV GAG protein or fragment thereof linked to the rhCDKL5 protein can be derived from a wild type HIV GAG protein comprising amino acid mutation selected from L31R, V35E, W36A or a combination thereof. In some embodiments, the HIV GAG protein or fragment thereof linked to the rhCDKL5 protein can be derived from a wild-type HIV GAG protein comprising amino acid mutations L31R, V35E and W36A. In some embodiments, the HIV GAG protein or fragment thereof linked to the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 12.

[0043] In some embodiments, the exosome trafficking tag linked rhCDKL5 protein comprises an affinity purification tag. In some embodiments, the affinity purification tag is linked to the exosome trafficking tag. In some embodiments, the affinity purification tag is linked to the C- terminus of the exosome trafficking tag. In some embodiments, the affinity purification tag is linked to the N-terminus of the exosome trafficking tag. In some embodiments, the affinity purification tag is selected from: (a) a polyhistidine tag; (b) a polyarginine tag; (c) a glutathione-S-transferase tag; (d) a DYKDDDDK (SEQ ID NO: 29) peptide tag; (e) a streptavidin-binding peptide tag; (f) a peptide tag comprising an amino acid sequence according to any one of SEQ ID NOs: 36 or 37; (g) a calmodulin binding peptide tag; (h) a maltose-binding tag; and (i) a cellulose-binding tag. In some embodiments, the affinity purification tag is a polyhistidine tag. In some embodiments, the polyhistidine tag is a 6xHIS tag (SEQ ID NO: 21).

[0044] In some embodiments, the method further comprises purifying the rhCDKL5 protein (e.g., exosome trafficking tag linked rhCDKL5 protein) from the supernatant using: (a) a Ni- NTA chromatography resin; (b) a cross-linked dextran chromatography resin; (c) an immobilized glutathione resin; (d) an anti-DYKDDDDK (SEQ ID NO: 29) peptide tag antibody resin; (e) a streptavidin resin; (f) a biotin resin; (g) an immobilized chitin resin; (h) a cellulose resin; (i) a maltose resin; or (j) a calmodulin resin. In some embodiments, the method further comprises purifying the rhCDKL5 protein from the supernatant using a Ni-NTA chromatography resin.

[0045] In some embodiments, the affinity purification tag and / or the exosome trafficking tag is linked to the N-terminus or C-terminus of the rhCDKL5 protein by a Tobacco etch virus (TEV) protease cleavage site linker. In some embodiments, the exosome trafficking tag and the affinity purification tag are linked to the N-terminus of the rhCDKL5 protein by a Tobacco etch virus (TEV) protease cleavage site linker.

[0046] In some embodiments, the rhCDKL5 comprises the exosome trafficking tag linked to the affinity purification tag, and the affinity purification tag is linked to the N-terminus of the rhCDKL5 protein by a Tobacco etch virus (TEV) protease cleavage site linker. In some embodiments, the rhCDKL5 comprises the exosome trafficking tag linked to the affinity purification tag, and the affinity purification tag is linked to the C-terminus of the rhCDKL5 protein by a Tobacco etch virus (TEV) protease cleavage site linker. In some embodiments, the exosome trafficking tag is linked by a linker peptide to the affinity purification tag.

[0047] In some embodiments, the rhCDKL5 protein comprises an N-terminal linked exosome trafficking tag and affinity purification tag, wherein the exosome trafficking tag is an HIV GAG protein or a fragment thereof and the affinity purification tag is a 6xHIS tag (SEQ ID NO: 21), wherein the exosome trafficking tag and the affinity purification tag are linked by a linker peptide, and wherein the affinity purification tag is linked to the N-terminus of the rhCDKL5 protein by a TEV protease cleavage site. In some embodiments, the rhCDKL5 protein comprises an HIV GAG protein or a fragment thereof and a 6xHIS tag (SEQ ID NO: 21) linked by a linker peptide, and wherein the 6xHIS tag (SEQ ID NO: 21) is linked to the N- terminus of the rhCDKL5 protein by a TEV protease cleavage site linker. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 12. In some embodiments, the HIV GAG protein or a fragment thereof comprises an amino acid sequence according to SEQ ID NO: 12. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 13. In some embodiments, the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 13.

[0048] In some embodiments, the method comprises expressing the exosome trafficking tag linked rhCDKL5 protein in the host cell culture, wherein the host cell culture is maintainedunder conditions adequate to express the rhCDKL5 protein for a duration up to 7 days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days). In some embodiments, the method comprises separating the supernatant comprising the exosomes comprising the expressed rhCDKL5 protein from the host cell culture is repeated or continuous throughout the duration. In some embodiments, the method comprises separating the supernatant comprising the rhCDKL5 protein (e.g., exosome trafficking tag linked rhCDKL5 protein) from the host cell culture and the separating is repeated or continuous throughout the duration of up to 7 days. In some embodiments, the method comprises separating the supernatant comprising the rhCDKL5 protein from the host cell culture every 1 day for up to 7 days. In some embodiments, the method comprises separating the supernatant comprising the rhCDKL5 protein from the host cell culture on the 2nd, 4th, and / or 6thday. In some embodiments, the method comprises separating the supernatant comprising the rhCDKL5 protein from the host cell culture on the 1st, 3rd, 5thand / or 7thday.

[0049] In some embodiments, maintaining the host cell culture under conditions adequate to express the rhCDKL5 protein comprises maintaining the culture at about 32oC to about 37oC (about 32oC to about 33oC, about 33oC to about 34oC, about 34oC to about 35oC, about 35oC to about 36oC, about 36oC to about 37oC, about 32oC to about 34oC, about 33oC to about 36oC, about 34oC to about 37oC, and all integers including and between 32oC and 37oC). In some embodiments, the host cell culture is maintained at about 37oC.

[0050] The present disclosure also provides use of any of the disclosed rhCDKL5 proteins or a fragment thereof produced by any of the methods disclosed herein in an analytical method for detecting a CDKL5-specific antibody in a sample. In some embodiments, the antibody is an anti-drug-antibody (ADA) raised in a subject administered a therapeutic CDKL5 gene or protein. In some embodiments, the analytical method is any one of a colorimetric method, enzyme immunoassay (EIA), radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLIA), and a counting immunoassay (CIA).

[0051] In some embodiments, the analytical method comprises: (a) immobilizing the rhCDKL5 protein or a fragment thereof to a solid surface; (b) contacting the immobilized rhCDKL5 protein with the sample for an amount of time sufficient for binding of the immobilized rhCDKL5 protein to the CDKL5-specific antibody, if present in the sample, to form a bipartite complex of the rhCDKL5 protein and the CDKL5-specific antibody; (c) contacting the bipartite complex with a detection reagent that binds specifically to the CDKL5-specific antibody and not the rhCDKL5 protein, for an amount of time to form a tripartite complex of the detection reagent, the rhCDKL5 protein, and the CDKL5-specific antibody, wherein the detection reagent comprises a detectable moiety that generates a detectable signal; and (d) detecting the signal generated in step (c), wherein presence of the detectable signal indicates presence of CDKL5-specific antibody in the sample, and absence of the detectable signal indicates absence of CDKL5-specific antibody in the sample.

[0052] In some embodiments, the rhCDKL5 protein or fragment thereof is immobilized (e.g., directly or indirectly) to the solid surface by covalent crosslinking to the solid surface. In some embodiments, the rhCDKL5 protein or fragment thereof is immobilized to the solid surface by an anti-CDKL5 antibody bound to the solid surface or by covalent crosslinking to the solid surface. In some embodiments, the solid surface is polyethylene, polyacrylamide, polystyrene, agarose, glass, or silicone rubber.

[0053] In some embodiments, the analytical method further comprises: i. contacting the immobilized rhCDKL5 protein of step (a) with a coating / blocking buffer before proceeding with step (b); ii. contacting the bipartite complex of step (b) with a wash buffer before proceeding with step (c); iii. contacting the tripartite complex of step (c) with a wash buffer before proceeding with step (d); iv. contacting the tripartite complex of step (c) with a detection solution before proceeding with step (d); or v. a combination thereof.

[0054] In some embodiments, the detectable moiety is any one of an enzyme, a fluorophore, and a radioisotope. In some embodiments, the detectable signal is any one of a colorimetric signal, a fluorescent signal, and radiation. In some embodiments, the detectable moiety is an enzyme. In some embodiments, the detectable moiety is an enzyme and step (c) further comprises contacting the tripartite complex with a substrate for generating the detectable signal. In some embodiments, the enzyme is any one of a horse radish peroxidase, an alkaline phosphatase, a galactosidase, an acetylcholinesterase, or a catalase. In some embodiments, the fluorophore is any one of fluorescein isothiocyanate (FITC), phycoerythrin (PE) or a conjugate thereof, an Alexa Fluor®dye, a rhodamine dye, a Cyanine-based dye, an Orange G-dye, and sulforhodamine 101 acid chloride dye (Texas red). In some embodiments, the radioisotope is any one of90Y,111In,177Lu,99mTc123I,125I, and131I. In some embodiments, the detection reagent is an antibody or a fragment thereof.

[0055] In some embodiments, the rhCDKL5 protein or a fragment thereof to be immobilized to the solid surface is a rhCDKL5 protein produced by any of the methods disclosed herein. In some embodiments, the rhCDKL5 protein is a biotinylated rhCDKL5 protein. In some embodiments, the biotinylated rhCDKL5 protein is immobilized by contacting on a solid surface coated with streptavidin.

[0056] In some embodiments, the detection reagent is a rhCDKL5 protein produced by any of the methods disclosed herein. In some embodiments, the analytical method for detecting a CDKL5-specific antibody in a sample further comprises a step of contacting the rhCDKL5 protein with a partner 2 protein or a fragment thereof (e.g., SpyCatcher protein or a protein comprising an amino acid sequence of any one of SEQ ID NOs: 33-35) labelled with Ruthenium (Ru), before contacting the bipartite complex with a detection reagent.

[0057] The present disclosure also provides use of the rhCDKL5 protein produced by the method of the present disclosure, or a fragment thereof, in an analytical method of quantitatively measuring an amount of CDKL5 present in a sample solution. In some embodiments, the analytical method is any one of a colorimetric method, enzyme immunoassay (EIA), radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLIA), and counting immunoassay (CIA). In some embodiments, the sample is a tissue homogenate, a cell lysate, or other biological fluid from a subject. In some embodiments, the analytical method (for quantitative measuring CDKL5 amount) comprises: i. contacting the sample solution with a CDKL5 capture reagent and a CDKL5 detection reagent, and detecting a sample signal from the CDKL5 detection reagent in the sample solution; ii. measuring the sample signal; iii. contacting one or more reference solutions, each comprising a known amount of the rhCDKL5 protein, with the CDKL5 capture reagent and the CDKL5 detection reagent, and detecting one or more comparator signals from the CDKL5 detection reagent in each of the one or more solutions; iv. measuring the one or more comparator signals; and v. comparing the sample signal with the one or more comparator signals to quantitatively determine the concentration of CDKL5 in the sample solution. In some embodiments, the CDKL5 detection reagent from step i is different from the CDKL5 detection reagent used in step iii. In some embodiments, the CDKL5 detection reagent is an antibody or a fragment thereof.

[0058] In some embodiments, the CDKL5 capture reagent has binding specificity for a CDKL5 in the sample solution and for the rhCDKL5 protein or a fragment thereof in the standard solution or reference solution. In some embodiments, the CDKL5 capture reagent is anantibody or a fragment thereof. In some embodiments, the CDKL5 capture reagent is immobilized to a solid surface. In some embodiments, the CDKL5 capture reagent is immobilized by a protein A, protein G or protein L or a combination thereof, to a solid surface. In some embodiments, the CDKL5 capture reagent is immobilized by a protein A to a solid surface. In some embodiments, the CDKL5 detection reagent binds specifically to a CDKL5 in the sample solution and the rhCDKL5 protein or a fragment thereof in the standard solution (or reference solution) but does not bind to the CDKL5 capture reagent. In some embodiments, detecting a signal from the CDKL5 detection reagent comprises contacting the CDKL5 detection reagent with a secondary detection reagent that specifically binds the CDKL5 detection reagent and not the CDKL5 capture reagent. In some embodiments, the secondary detection reagent is an antibody or a fragment thereof. In some embodiments, the secondary detection reagent comprises a reporter moiety. In some embodiments, the reporter moiety is any one of a horse radish peroxidase, an alkaline phosphatase, a galactosidase, an acetylcholinesterase, and a catalase.

[0059] In some embodiments, the analytical method further comprises contacting the sample solution and the one or more reference solutions comprising known amounts of the rhCDKL5 protein with a washing solution before measuring the sample signal. In some embodiments, the sample is from a subject suffering from or suspected of having a CDKL5 deficiency disorder, CDKL5 encephalopathy, CDKL5-related epilepsy, CDKL5-related epileptic encephalopathy, early infantile epileptic encephalopathy 2. In some embodiments, the subject has been administered or is to be administered a therapeutic CDKL5 gene or protein. In some embodiments, the sample is a tissue homogenate, a cell lysate, or other biological fluid from a subject.

[0060] The present disclosure also provides a kit for detecting CDKL5-specific antibody in a sample, comprising: (a) a recombinant human CDKL5 (rhCDKL5) protein or a fragment thereof produced by any of the methods of the present disclosure; and (b) a detection reagent that specifically binds to the CDKL5-specific antibody but not to the rhCDKL5 protein or fragment thereof, wherein the detection reagent comprises a detectable moiety. In some embodiments, the kit for detecting CDKL5 specific antibody in a sample further comprises: i. a solid surface; ii. a coating / blocking solution; iii. a washing solution; iv. a detection solution; or v. a combination thereof.

[0061] In some embodiments of the kit of the present disclosure, the rhCDKL5 protein or fragment thereof is immobilized to the solid surface. In some embodiments, the rhCDKL5 protein or a fragment thereof to be immobilized to the solid surface is a rhCDKL5 protein of the present disclosure. In some embodiments, the rhCDKL5 protein is a biotinylated rhCDKL5 protein. In some embodiments, the biotinylated rhCDKL5 protein is immobilized on a solid surface coated with streptavidin.

[0062] In some embodiments of the kit of the present disclosure, the detection reagent is a rhCDKL5 protein of the present disclosure. In some embodiments, the kit for detecting CDKL5 specific antibody in a sample of the present disclosure comprises a partner 2 protein (SpyCatcher protein or a protein comprising an amino acid sequence of any one of SEQ ID NOs: 33-35) labeled with Ruthenium (Ru) for forming a complex with the rhCDKL5 protein, before contacting the bipartite complex with a detection reagent.

[0063] In some embodiments, the kit for detecting CDKL5 specific antibody in a sample of the present disclosure, further comprises an anti-CDKL5 antibody or a crosslinker for immobilizing the rhCDKL5 protein or fragment thereof to the solid surface. In some embodiments, the solid surface is coated with protein A agarose. In some embodiments, the solid surface is any one of polyethylene, polyacrylamide, polystyrene, agarose, glass, and silicone rubber. In some embodiments, the detectable moiety is any one of an enzyme, a fluorophore, and a radioisotope. In some embodiments, the detectable moiety generates a detectable signal which is any one of a colorimetric signal, a fluorescent signal, and radiation. In some embodiments, the detectable moiety is an enzyme, and the kit further comprises a substrate that generates a detectable signal upon reacting with the enzyme. In some embodiments, the enzyme is any one of a horse radish peroxidase, an alkaline phosphatase, a galactosidase, an acetylcholinesterase, and a catalase. In some embodiments, the fluorophore is any one of fluorescein isothiocyanate (FITC), phycoerythrin (PE) or a conjugate thereof, an Alexa Fluor®dye, a rhodamine dye, a Cyanine-based dye, an Orange G-dye, and sulforhodamine 101 acid chloride dye. In some embodiments, the radioisotope is any one of90Y,111In,177Lu,99mTc123I,125I, and131I. In some embodiments, the detection reagent is an antibody or a fragment thereof.

[0064] The present disclosure also provides a kit for quantitatively measuring an amount of CDKL5 present in a sample solution comprising: (a) a CDKL5 capture reagent; (b) a CDKL5 detection reagent; and (c) a standard solution comprising a rhCDKL5 protein produced by anyof the methods of the present disclosure or a fragment thereof, or a lyophilized reagent for preparing said standard solution.

[0065] In some embodiments of the kit for quantitatively measuring an amount of CDKL5 present in a sample solution of the present disclosure, the CDKL5 detection reagent binds specifically to CDKL5 in the sample and the rhCDKL5 protein or a fragment thereof in the standard solution or reference solution. In some embodiments, the CDKL5 detection reagent is an antibody or a fragment thereof that specifically binds to rhCDKL5 protein.

[0066] In some embodiments, the kit for quantitatively measuring an amount of CDKL5 present in a sample solution of the present disclosure further comprises: i. a solid surface; ii. a coating solution; iii. a washing solution; iv. a detection solution; or v. a combination thereof. In some embodiments, the CDKL5 capture reagent binds specifically to CDKL5 in the sample and the rhCDKL5 protein or a fragment thereof in the standard solution or reference solution. In some embodiments, the CDKL5 capture reagent is an antibody or a fragment thereof. In some embodiments, the CDKL5 capture reagent is an antibody or a fragment thereof that specifically binds to CDKL5 at a site that is different from the binding site of the CDKL5 detection reagent. In some embodiments, the CDKL5 capture reagent is immobilized to the solid surface. In some embodiments, the CDKL5 capture reagent is immobilized by protein A, protein G, protein L or a combination thereof, to the solid surface. In some embodiments, the CDKL5 capture reagent is immobilized by protein A to the solid surface. In some embodiments, the CDKL5 detection reagent binds specifically to CDKL5 in the sample and the rhCDKL5 protein or a fragment thereof in the standard solution or reference solution but does not bind to the CDKL5 capture reagent. In some embodiments, the CDKL5 detection reagent is an antibody or a fragment thereof. In some embodiments, the CDKL5 detection reagent is different from the CDKL5 capture reagent. In some embodiments, the kit further comprises a secondary detection reagent that specifically binds the CDKL5 detection reagent and not the CDKL5 capture reagent. In some embodiments, the secondary detection reagent is an antibody or a fragment thereof. In some embodiments, the secondary detection reagent comprises a reporter moiety. In some embodiments, the reporter moiety is any one of a horse radish peroxidase, an alkaline phosphatase, a galactosidase, an acetylcholinesterase, and a catalase.

[0067] The present disclosure also provides a method of producing a full-length, non- denatured, soluble, and tagless rhCDKL5 protein, wherein the method comprises the steps of:a) providing a rhCDKL5 protein linked to one or more intracellular expression tags or an exosome trafficking tag linked by a TEV cleavage site linker to the N- and C-terminus of the rhCDKL5 protein, produced by any of the method disclosed herein; b) incubating the rhCDKL5 protein with an effective amount of a TEV protease to remove the TEV protease cleavage site linker and generate a reaction mixture comprising a tagless recombinant human CDKL5 protein and impurities comprising cleaved one or more intracellular expression peptide tags or one or more exosome trafficking tags and partially cleaved rhCDKL5 proteins; c) applying the reaction mixture to an immobilized-metal affinity chromatography (IMAC) resin to generate a flowthrough comprising the impurities; and d) applying an elution buffer to the IMAC resin to generate an eluent comprising the tagless recombinant human CDKL5 protein.

[0068] In some embodiments, the method further comprises removing any remaining impurities and purifying the tagless CDKL5 protein from the eluent by passing the eluent through a hydrophobic interaction chromatography resin to generate a second eluent comprising the tagless CDKL5 protein. In some embodiments, the method further comprises passing the eluent comprising the tagless recombinant human CDKL5 protein through a hydrophobic interaction chromatography resin to generate a second eluent comprising the tagless recombinant human CDKL5 protein

[0069] The present disclosure also provides a method of expressing CDKL5 protein in a subject in need thereof, by administering to the subject a therapeutically effective amount of a human CDKL5 protein generated by any of the methods of the present disclosure. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human, a non- human primate or a rodent. In some embodiments, the method further comprises, detecting the presence of human CDKL5 in the subject by: i) obtaining a sample from the subject; ii) contacting the sample with one or more proteolytic enzyme(s) for an amount of time sufficient to generate one or more peptide fragments; and iii) detecting the presence of a human specific CDKL5 sequence in the one or more peptide fragments of step ii). In some embodiments, the one or more proteolytic enzyme(s) is selected from a group consisting of trypsin, chymotrypsin, Lys-C endoproteinase, AspN endoproteinase, GluC endoproteinase, chymotrypsin, and proteinase K. In some embodiments, the method comprises contacting the sample with Lys-C endoproteinase and chymotrypsin.

[0070] In some embodiments, the method comprises detecting of the presence of a human specific CDKL5 sequence in the one or more peptide fragments by any one of a massspectrometry (MS), colorimetric method, enzyme immunoassay (EIA), radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLIA), and counting immunoassay (CIA). In some embodiments, the mass spectrometry is a liquid chromatography- mass spectrometry (LC-MS). In some embodiments, the LC-MS comprises any one of adsorption chromatography, partition chromatography, ion-exchange chromatography, size- exclusion chromatography, and affinity chromatography. In some embodiments, the human- specific CDKL5 sequence comprises an amino acid sequence according to SEQ ID NO: 28.

[0071] The present disclosure also provides anti-CDKL5 antibodies or CDKL5-binding fragments thereof. In some embodiments, the anti-CDKL5 antibodies or fragments thereof of the present disclosure bind the non-catalytic / non-kinase domain of a human CDKL5 protein. In some embodiments, the anti-CDKL5 antibodies or fragments thereof of the present disclosure bind the catalytic / kinase domain of a human CDKL5 protein.

[0072] In some embodiments, the anti-CDKL5 antibody or fragment thereof binds to the kinase domain of the CDKL5 protein. In some embodiments, the anti-CDKL5 antibody or fragment thereof binds to the non-catalytic / non-kinase domain of the CDKL5 protein.

[0073] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 42, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 43 and a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 44.

[0074] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 45, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 46 and a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 47.

[0075] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 48, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 49 and a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 50.

[0076] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 51, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 52 and a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 53.

[0077] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 54, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 55 and a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 56.

[0078] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 57, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 58 and a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 59.

[0079] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a LCDR1 comprising an amino acid sequence according to SEQ ID NO: 60, a LCDR2 comprising an amino acid sequence of LVS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 62.

[0080] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a LCDR1 comprising an amino acid sequence according to SEQ ID NO: 63, a LCDR2 comprising an amino acid sequence of KVS and a LCDR3 according to SEQ ID NO: 65.

[0081] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a LCDR1 comprising an amino acid sequence according to SEQ ID NO: 66, a LCDR2 comprising an amino acid sequence of KVS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 67.

[0082] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a LCDR1 comprising an amino acid sequence according to SEQ ID NO: 68, a LCDR2 comprising an amino acid sequence of LVS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 69.

[0083] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a LCDR1 comprising an amino acid sequence according to SEQ ID NO: 70, a LCDR2 comprising an amino acid sequence of WAS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 72.

[0084] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a LCDR1 comprising an amino acid sequence according to SEQ ID NO: 73, a LCDR2comprising an amino acid sequence of QVS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 75.

[0085] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 42, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 43, a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 44, LCDR1 comprising an amino acid sequence according to SEQ ID NO: 60, a LCDR2 comprising an amino acid sequence of LVS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 62.

[0086] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 45, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 46, a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 47, LCDR1 comprising an amino acid sequence according to SEQ ID NO: 63, a LCDR2 comprising an amino acid sequence of KVS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 65.

[0087] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 48, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 49, a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 50, LCDR1 comprising an amino acid sequence according to SEQ ID NO: 66, a LCDR2 comprising an amino acid sequence of KVS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 67.

[0088] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 51, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 52, a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 53, LCDR1 comprising an amino acid sequence according to SEQ ID NO: 68, a LCDR2 comprising an amino acid sequence of LVS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 69.

[0089] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 54, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 55, a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 56, LCDR1 comprising an amino acid sequenceaccording to SEQ ID NO: 70, a LCDR2 comprising an amino acid sequence of WAS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 72.

[0090] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 57, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 58, a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 59, LCDR1 comprising an amino acid sequence according to SEQ ID NO: 73, a LCDR2 comprising an amino acid sequence of QVS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 75.

[0091] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence of any one of SEQ ID Nos: 76-81.

[0092] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 76. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 77. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 78. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 79. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 80. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 81.

[0093] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a light chain variable region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence of any one of SEQ ID Nos: 82-87.

[0094] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 82. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 83. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a light chain variableregion comprising an amino acid sequence according to SEQ ID NO: 84. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 85. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 86. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 87.

[0095] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 76, and a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 82. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 77, and a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 83. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 78, and a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 84. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 79, and a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 85. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 80, and a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 86. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 81, and a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 87.

[0096] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence of any one of SEQ ID Nos: 88-93.

[0097] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain comprising an amino acid sequence according to SEQ ID NO: 88. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain comprising an amino acid sequence according to SEQ ID NO: 89. In some embodiments, the anti-CDKL5 antibodyor fragment thereof comprises a heavy chain comprising an amino acid sequence according to SEQ ID NO: 90. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain comprising an amino acid sequence according to SEQ ID NO: 91. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain comprising an amino acid sequence according to SEQ ID NO: 92. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain comprising an amino acid sequence according to SEQ ID NO: 93.

[0098] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a light chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence of any one of SEQ ID Nos: 94-99. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a light chain comprising an amino acid sequence according to SEQ ID NO: 94. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a light chain comprising an amino acid sequence according to SEQ ID NO: 95. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a light chain comprising an amino acid sequence according to SEQ ID NO: 96. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a light chain comprising an amino acid sequence according to SEQ ID NO: 97. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a light chain comprising an amino acid sequence according to SEQ ID NO: 98. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a light chain comprising an amino acid sequence according to SEQ ID NO: 99.

[0099] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain comprising an amino acid sequence according to SEQ ID NO: 88, and a light chain comprising an amino acid sequence according to SEQ ID NO: 94. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain comprising an amino acid sequence according to SEQ ID NO: 89, and a light chain comprising an amino acid sequence according to SEQ ID NO: 95. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain comprising an amino acid sequence according to SEQ ID NO: 90, and a light chain comprising an amino acid sequence according to SEQ ID NO: 96. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain comprising an amino acid sequence according to SEQ ID NO: 91, and a light chain comprising an amino acid sequence according to SEQ ID NO: 97. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain comprising an amino acid sequenceaccording to SEQ ID NO: 92, and a light chain comprising an amino acid sequence according to SEQ ID NO: 98. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises a heavy chain comprising an amino acid sequence according to SEQ ID NO: 93, and a light chain comprising an amino acid sequence according to SEQ ID NO: 99.

[0100] The present disclosure also provides one or more nucleic acid(s) encoding the anti- CDKL5 antibody or fragment thereof disclosed here. In some embodiments, the one or more nucleic acid(s) encodes a heavy chain variable region and comprises a nucleotide sequence that is at least 95% identical to a nucleotide sequence of any one of SEQ ID Nos: 100-105.

[0101] In some embodiments, the one or more nucleic acid(s) encodes a heavy chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 100. In some embodiments, the one or more nucleic acid(s) encodes a heavy chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 101. In some embodiments, the one or more nucleic acid(s) encodes a heavy chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 102. In some embodiments, the one or more nucleic acid(s) encodes a heavy chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 103. In some embodiments, the one or more nucleic acid(s) encodes a heavy chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 104. In some embodiments, the one or more nucleic acid(s) encodes a heavy chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 105.

[0102] In some embodiments, the one or more nucleic acid(s) encodes a light chain variable region and comprises a nucleotide sequence that is at least 95% identical to a nucleotide sequence of any one of SEQ ID Nos: 106-111.

[0103] In some embodiments, the one or more nucleic acid(s) encodes a light chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 106. In some embodiments, the one or more nucleic acid(s) encodes a light chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 107. In some embodiments, the one or more nucleic acid(s) encodes a light chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 108. In some embodiments, the one or more nucleic acid(s) encodes a light chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 109. In some embodiments, the one or more nucleic acid(s) encodes a light chainvariable region and comprises a nucleotide sequence according to SEQ ID NO: 110. In some embodiments, the one or more nucleic acid(s) encodes a light chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 111.

[0104] In some embodiments, the one or more nucleic acid(s) comprises a nucleotide sequence according to SEQ ID NO: 100 encoding a heavy chain variable region, and a nucleotide sequence according to SEQ ID NO: 106 encoding a light chain variable region.

[0105] In some embodiments, the one or more nucleic acid(s) comprises a nucleotide sequence according to SEQ ID NO: 101 encoding a heavy chain variable region, and a nucleotide sequence according to SEQ ID NO: 107 encoding a light chain variable region.

[0106] In some embodiments, the one or more nucleic acid(s) comprises a nucleotide sequence according to SEQ ID NO: 102 encoding a heavy chain variable region, and a nucleotide sequence according to SEQ ID NO: 108 encoding a light chain variable region.

[0107] In some embodiments, the one or more nucleic acid(s) comprises a nucleotide sequence according to SEQ ID NO: 103 encoding a heavy chain variable region, and a nucleotide sequence according to SEQ ID NO: 109 encoding a light chain variable region.

[0108] In some embodiments, the one or more nucleic acid(s) comprises a nucleotide sequence according to SEQ ID NO: 104 encoding a heavy chain variable region, and a nucleotide sequence according to SEQ ID NO: 110 encoding a light chain variable region.

[0109] In some embodiments, the one or more nucleic acid(s) comprises a nucleotide sequence according to SEQ ID NO: 105 encoding a heavy chain variable region, and a nucleotide sequence according to SEQ ID NO: 111 encoding a light chain variable region.

[0110] In some embodiments, the present disclosure provides one or more expression vector(s) comprising one or more nucleic acid(s) disclosed herein. In some embodiments, the expression vector comprises any one of a plasmid, a viral vector, and a cosmid.

[0111] These and other aspects and features are described in the following sections of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0112] The invention can be more completely understood with reference to the following drawings.

[0113] FIGs. 1A-1C are illustrative diagrams depicting structural and functional domains of a full-length human cyclin domain kinase 5 (hCDKL5) protein. FIG. 1A depicts the spatial arrangement of the N-terminal protein kinase domain, intrinsic disordered domains (Disordered), ATP binding site, Proton Acceptor ACT site, and Phosphoserine modified residue (MOD RES), are as indicated. FIG.1B depicts the predicted 3D structure of hCDKL5 with the protein kinase domain (labeled as “Kinase Domain (non-shaded)” in the figure), the intrinsic disordered protein (IDP) sequence containing Regulatory domain (labeled as “IDP (shaded)” in the figure), and nuclear localization sequences (labeled as “Nuclear Localization Sequences (Black)” in the figure). FIG. 1C depicts the predicted 2D structure of hCDKL5 (labeled as “CDKL5” in the figure) with the protein kinase domain (labeled as “Catalytic domain” in the figure), the nuclear localization sequences (labeled “NLS1” and “NLS2” in the figure), and Regulatory domain, are as indicated. FIG.1C discloses SEQ ID NOs: 112 and 3, respectively, in order of appearance.

[0114] FIGs.2A-2F show illustrative diagrams depicting alternative configurations of rhCDKL5 proteins (CDKL5 protein kinase domain indicated with light shading and CDKL5 IDP labeled as “disordered domain” indicated with dark shading) having solubility, secretion or exosome trafficking, and detection and / or affinity tags. FIGs.2A-2B are diagrams showing two different configurations of soluble rhCDKL5 protein with different orientations of MBP or GFP tags linked via a Tobacco Etch Virus (TEV) linker to the N- or C-terminus of the rhCDKL5 protein of the disclosure, as indicated. FIGs. 2C-2E are diagrams showing three different designs of a secretable rhCDKL5 protein with any one of an immunoglobulin light chain secretion signal tag (Light chain SS) (FIG.2C), an immunoglobulin heavy chain signal sequence (Heavy chain SS) (FIG.2D), or a hemagglutinin signal sequence (HA SS) (FIG.2E) linked to the N-terminus, and a polyhistidine (6xHIS tag (SEQ ID NO: 21)) affinity tag linked to the C-terminus of the rhCDKL5 protein of the disclosure, as indicated. FIG.2F is a diagram showing a configuration of an exosome trafficable rhCDKL5 protein comprising an exosome trafficking tag (GAG protein or a fragment thereof) connected by a linker (GGSTSSEFCSRRYRGPGIHRPVAT, SEQ ID NO: 113) to a 6xHIS (SEQ ID NO: 21) affinity tag which is linked via a TEV linker to the N-terminus of the rhCDKL5 protein of the disclosure, as indicated. FIGs.2C-2F all disclose “6xHis” as SEQ ID NO: 21.

[0115] FIG.3 is a schematic diagram showing three different approaches of producing an intracellularly expressed, secreted or exosome trafficked full-length rhCDKL5 protein, asindicated. The rhCDKL5 protein can be linked to specific tags for increasing solubility / expression, secretion signal tags or exosome trafficking tag and / or detection / affinity tags of FIGs.2A-2F, as indicated. FIG.3 discloses “6xHIS” as SEQ ID NO: 21.

[0116] FIGs. 4A-4C show increased solubility and expression of the intracellular expression tag containing rhCDKL5 protein of the present disclosure. FIG. 4A shows fluorescence microscopy images depicting intracellular expression of rhCDKL5 in Expi293 cells at 48 hours post-transfection with a plasmid encoding a rhCDKL5 protein with either a solubility MBP-TEV tag linked to the N-terminus and a detection GFP-TEV tag linked to the C-terminus (MBP-TEV-CDKL5-TEV-GFP), or a solubility GFP-TEV tag linked to the N- terminus and a detection tag MBP-TEV tag linked to the C-terminus (GFP-TEV-CDKL5-TEV- MBP), as indicated. FIG. 4B depicts SDS-PAGE images (images titled “SDS-PAGE”) for detecting total protein levels and Western blot images (images titled “anti CDKL5” and “anti MBP”) showing the presence of rhCDKL5 detected by anti-CDKL5 antibody and anti-MBP antibody, as indicated, in lysate derived from Expi293 cells 48 hours post-transfection with plasmid construct encoding the MBP-TEV-CDKL5-TEV-GFP rhCDKL5 protein. FIG. 4C depicts an SDS-PAGE image (image titled “SDS-PAGE”) for detecting total protein levels and a Western blot image (image titled “anti CDKL5”) showing the presence of rhCDKL5 detected by anti-CDKL5 antibody as indicated, in lysate derived from Expi293 cells 48 hours post- transfection with plasmid construct encoding the MBP-TEV-CDKL5-TEV-GFP rhCDKL5 protein in presence or absence of 150 nM BafA1 and GFP-TEV-CDKL5-TEV-MBP rhCDKL5 protein.

[0117] FIGs. 5A-5D show strategies for improving solubility and expression of the rhCDKL5 produced from cells transfected with GFP-TEV-CDKL5-TEV-MBP rhCDKL5 protein encoding plasmid construct. FIG. 5A depicts SDS-PAGE image (left panel) for detecting total protein levels and Western blot image probed with anti-CDKL5 antibody (right panel titled “PVDF probed with α-CDKL5 primary Ab”) showing the presence of rhCDKL5, in lysate derived from Expi293 cells at 48 hours and 72 hours post-transfection with plasmid construct encoding the GFP-TEV-CDKL5-TEV-MBP protein at 37oC or 32oC in presence or absence of enhancer, as indicated. FIG.5B depicts SDS-PAGE image detecting total protein levels (left panel titled “4-20% Criterion TGX Stain-free gel (Reduced / Denatured”) and Western blot image probed with anti-MBP antibody showing the presence of rhCDKL5 (right panel titled “PVDF probed with α-MBP primary Ab”), in lysates derived by lysis of Expi293cells at 72 hours post-transfection with plasmid construct encoding the GFP-TEV-CDKL5- TEV-MBP protein. Lanes loaded with whole cell lysates in presence (Pierce) or absence (No Inh) of protease inhibitor (W1 and W2); cell lysate derived by lysis of cells in lysis buffer in presence (Pierce) or absence (No Inh) of protease inhibitor, with incubation periods of 0 min, 15 min, 30 min, 60 min or overnight, and insoluble fractions in cell pellet (P1 and P2), are as indicated. FIG. 5C is an MBPTrap affinity chromatography step elution profile of the rhCDKL5 protein purified from the soluble fraction obtained prepared from Expi293 cells transfected with GFP-TEV-CDKL5-TEV-MBP protein encoding plasmid construct for 72 hours post-transfection. FIG. 5D depicts SDS-PAGE image detecting total protein levels in eluent fractions (left panel titled “4-20% Criterion TGX Stain-free gel (Reduced / Denatured”) and Western blot image probed with anti-MBP antibody showing the presence of rhCDKL5 in eluent fraction 3 (right panel titled “Anti-MBP WB”), of the MBPTrap affinity chromatography run of FIG.5C. Lanes loaded with whole cell lysate (W), soluble extract (S), insoluble fraction (I), FPLC load (L), Flow through (FT), wash (W), and FPLC elution fraction (2-6), are as indicated.

[0118] FIG.6A-6F show evaluating parameters and methods of expression, processing and purification of the intracellular expression tag linked rhCDKL5 protein (GFP-TEV- CDKL5-TEV-MBP), of the present disclosure. FIG. 6A is a Western blot of reduced lysates obtained by lysis and sonication from Expi293 cells expressing the GFP-TEV-CDKL5-TEV- MBP protein and probed with anti-MBP antibody. The lanes loaded with whole cell lysate, insoluble fraction, soluble fraction (soluble extract), and filtered soluble fraction (0.45 um filtered soluble extract), are as indicated. FIG. 6B shows SDS-PAGE gel image (left panel) and an anti-MBP antibody probed Western blot (right panel), of reduced lysates obtained by lysis and homogenization from Expi293 cells expressing the GFP-TEV-CDKL5-TEV-MBP protein. The marker lane (M), mock control (Mk), cells resuspended in lithium dodecyl sulfate (LDS) buffer (-1), whole cell lysates (W1 and W2), are as indicated. FIG.6C is an anti-MBP antibody probed Western blot of reduced lysates obtained by lysis of cells expressing the GFP- TEV-CDKL5-TEV-MBP protein using either a liquid microfluidizer (LM) or a liquid homogenizer (LH), as indicated. FIG.6D is an MBPTrap affinity chromatography step elution profile of the rhCDKL5 protein purified from lysate prepared by liquid homogenization of Expi293 cells transfected with GFP-TEV-CDKL5-TEV-MBP protein encoding plasmid construct for 72 hours post-transfection. FIG.6E is an anti-MBP antibody probed Western blotof lysates obtained by lysis of cells expressing the GFP-TEV-CDKL5-TEV-MBP protein using a liquid homogenizer (LH), under reduced (denoted as R in the image) and non-reducing condition (denoted as NR in the image), as indicated. FIG.6F is an anti-MBP antibody probed Western blot showing detection of rhCDKL5 produced by TEV cleavage of purified GFP- TEV-CDKL5-TEV-MBP protein, using 1500 IU / ml of TEV enzyme in 10 mM Tris-HCl, pH 8.5, at room temperature for 4 hours (4 hrs) or overnight (ON), as indicated.

[0119] FIG. 7 is an anti-CDKL5 antibody probed Western blot showing detection of CDKL5 separated from impurities by purification using an AEX column at pH of between 6.5- 7.0, post-TEV cleavage and tag removal from GFP-TEV-CDKL5-TEV-MBP protein purified from cell lysates. The detection of tagless CDKL5 in loading material (denoted as load in the image), flow through (denoted as FT in the image), wash, individual elution fractions (denoted as eluted fractions) and pooled post-cleavage CDKL5 eluent fractions (denoted as CIP in the image), are as indicated.

[0120] FIGs.8A-8C depict separating the full-length CDKL5 protein post enzymatic cleavage using immobilized metal affinity chromatography (IMAC) and further concentration and purification by hydrophobic interaction chromatography. FIG.8A is a Western blot probed with anti-CDKL5 antibody showing IMAC purification of fully cleaved CDKL5 protein from impurities (tag peptides and partially digested tagged CDKL5 proteins) at a pH of 8.0 and 8.5. The lanes corresponding to the marker (M), loading material (L), flowthrough (A6) and eluted fractions (B1-B4), are as indicated. FIG. 8B is a Western blot probed with anti-CDKL5 antibody showing hydrophobic butyl chromatography for purification of fully cleaved CDKL5 protein from impurities (tag peptides and partially digested tagged CDKL5 proteins). The lanes corresponding to the marker, loading material (Load), flowthrough (FT) and eluted fractions, are as indicated. FIG. 8C is a schematic depicting the steps of expression, isolation and purification of a tagless CDKL5 protein from an intracellular expression tagged rhCDKL5 protein expressed in a mammalian host cell culture, disclosed herein. The specific steps of expressing the rhCDKL5 protein in cells of host cell culture, lysis, capture step for extraction of the rhCDKL5 protein, TEV tag cleavage and further purification and polishing steps using IMAC and HIC purification, are as indicated.

[0121] FIG. 9 is a Western blot showing detection of CDKL5 in supernatants from cultures of Expi293 cells harvested at 1, 2, 3, 6, and 7 days after transfection with plasmids encoding the three rhCDKL5 linked to secretory signal peptide tags (light chain signalsequence -CDKL5-6xHIS (denoted as “LC SS” in the figure); heavy chain signal sequence - CDKL5-6xHis (denoted as “HC SS” in the figure)and Hemagglutinin signal sequence - CDKL5-6xHis) (denoted as “HA SS” in the figure) of the present disclosure, and a mock plasmid (same plasmid as encoding the three rhCDKL5 but without the CDKL5 coding sequence and denoted as “Mock” in the figure), as indicated.

[0122] FIG. 10 is a Western blot showing the expression level of rhCDKL5 obtained from exosomes in supernatants harvested from Expi293 cells harvested at 1, 2, 3, 6, and 7 days after transfection with mock plasmid (denoted as “1.” in the figure) and plasmid encoding the exosome trafficking tag linked rhCDKL5 protein (GAG-linker-6xHis-TEV-CDKL5) (denoted as “2.” in the figure of the present disclosure, as indicated.

[0123] FIGs.11A-11C show detection of pan-CDKL5 peptide (the peptide of SEQ ID NO: 41) in wild-type (WT) mouse brains digest. FIG.11A depict HPLC detection profiles pan CDKL5 peptide (the peptide of SEQ ID NO: 41) in digested brain tissue of a representative wild type mouse brain section (WT / P18 (Cdkl5+ / +)) and hemizygous Cdkl5- / ymice brain section, (HEMI / N18 (Cdkl5- / y)) as indicated. P16, P17, P18, P19 and P20 are sample numbers that correspond to slices of brain sections of wild type mouse brain, as indicated in the brain schematic in FIG.11B, and N12, N14, N18, N19 and N20 are sample numbers that correspond to slices of brain of Cdkl5- / y(HEMI) mouse brain. Peaks specific for the peptide of SEQ ID NO: 41 in WT and Cdkl5- / ymice, are indicated by boxes. FIG. 11B is a histogram plot representing the level of the peptide of SEQ ID NO: 41 detected in brain digests of each mice within group of WT and Cdkl5- / y(HEMI) mice. FIG.11C is a nanoflow liquid chromatography profile of the peptide of SEQ ID NO: 41 detected in WT (WT / P16-20) and Cdkl5- / y(HEMI / N12, 14, 18-20) mice brain digests.

[0124] FIGs. 12A-12C show dose dependent detection of CDKL5 in mouse brain. FIG. 12A is a schematic description of the process of cleaving proteins hemizygous Cdkl5- / y(HEMI) mouse brain lysate spiked with both rhCDKL5 and N15-labelled human CDKL5 (15N-CDKL5) and analyzed by nanoflow liquid chromatography. FIG. 12B depicts the nanoflow liquid chromatography detection profile of peptide of SEQ ID NO: 41 in rhCDKL5 (SEQ ID NO: 41 in rhCDKL5) and N15-labelled human CDKL5 (SEQ ID NO: 41 in 15N- CDKL5) spiked mouse brain tissue lysate, as indicated. FIG. 12C is a peak area ratio curve depicting the dose dependent detection of peptide of SEQ ID NO: 41 from rhCDKL5 spiked in increasing amounts into Cdkl5- / y brain (HEMI) mouse brain tissue (matrix) lysate or matrixfree buffer each containing a constant amount of spiked N15-labelled CDKL5 digested and analyzed, as indicated.

[0125] FIGs. 13A-13D show specific detection of human rhCDKL5 in mouse brain tissue lysate. FIG. 13A is a schematic description of the process of spiking native and N15 labeled recombinant hCDKL5 (15N-CDKL5) in rat brain lysate at a physiological level (spike to total protein ratio 1:100,000), and further cleaved and digested into peptides and analyzed by nanoflow liquid chromatography. FIG. 13B depicts the nanoflow liquid chromatography detection profile of human CDKL5 specific peptide of SEQ ID NO: 28 (denoted as SEQ ID NO: 28) and N15 labeled human CDKL5 specific peptide of SEQ ID NO: 28 (denoted as 15N- SEQ ID NO: 28) in digested native and N15 labeled recombinant hCDKL5 spiked rat brain lysate, as indicated. Peak specific for peptide of SEQ ID NO: 28 and N15 labeled peptide of SEQ ID NO: 28 are indicated by the oval. FIGs. 13C-13D depict nanoflow liquid chromatography detection profiles of N15labeled human specific peptide of SEQ ID NO: 28 (upper panels) (denoted as (15N-)SEQ ID NO: 28 (human specific), N15labeled peptide of SEQ ID NO: 40 (middle panels) (denoted as (15N-)SEQ ID NO: 40), N15labeled peptide of SEQ ID NO: 41 (lower panels) (denoted as (15N-)SEQ ID NO: 41), in native (FIG. 13C) or N15-labelled human CDKL5 (15N‐CDLK5) (FIG. 13D) spiked mouse brain tissue lysate, as indicated.

[0126] FIGs.14A-14D show strategies for a bridging assay for anti-CDKL5 antibody detection in samples. FIG. 14A is a schematic of a CDKL5 biotinylable protein showing an intracellular expression tagged CDKL5 biotinylable protein comprising a C-terminal peptide tag comprising an amino acid sequence according to SEQ ID NO: 14 (denoted as AviTAG), as indicated, for in vivo biotinylation. FIG. 14B is a schematic depicting the lysine (K) residue of SEQ ID NO: 14 that is biotinylated. FIG.14C is a schematic of a CDKL5 partner 1 protein showing an intracellular expression tagged CDKL5 partner 1 protein comprising an N-terminal peptide tag comprising an amino acid sequence according to any one of SEQ ID NOs: 30-32 or a fragment thereof, as indicated (SPY-Tag). CDKL5 partner 1 protein bind to a labeled “partner 2 protein” comprising an amino acid sequence according to any one of SEQ ID NOs: 33-35 or a fragment thereof. FIG. 14D is a schematic showing the process of using the biotinylated CDKL5 linked partner 1 protein and partner 2 protein that is Ru labeled, for capturing and detecting anti-CDKL5 antibody in a sample on a streptavidin coated solid surface, as indicated.

[0127] FIGs. 15A-15B are enzyme linked immunosorbent assay (ELISA) plots showing target antigen binding by serum isolated from mice on day 35 post-immunization with GFP-CDKL5-MBP immunogen (tagged CDKL5) either with alhydrogel / muramyl dipeptide (ALD-MDP) (Hu CDKL5a group) or Sigma Adjuvant System (SAS) (Hu CDKL5s group) as adjuvant. Preimmunization mouse sera was used as a control. Absorbance showing level of antigen binding to tagged CDKL5 (GFP-CDKL5-MBP) (FIG. 15A) and untagged CDKL5 protein (CDKL5) (FIG. 15B), is depicted on y-axis with respect to antiserum dilution, as depicted on x-axis. The antigen binding for antiserum from each mouse within the Hu CDKL5a group (denoted as Hu CDKL5a m1, D35, Hu CDKL5a m2, D35, and Hu CDKL5a m3, D35), Hu CDKL5s group (denoted as Hu CDKL5s m1, D35, Hu CDKL5s m2, D35, and Hu CDKL5s m3, D35), one mouse from HuCDKL5s group on day 21 (an earlier time point control) (denoted as Hu CDKL5s m1, D21), and pre-immune serum (denoted as Preimmune mouse sera), are as indicated.

[0128] FIGs. 16A-16B are ELISA plots showing target antigen binding by serum isolated from mice on day 63 post-immunization with GFP-CDKL5-MBP immunogen (tagged CDKL5) either with ALD-MDP (Hu CDKL5a group) or Sigma Adjuvant System (SAS) (Hu CDKL5s group) as adjuvant. Absorbance showing level of binding to untagged CDKL5 protein is depicted on y-axis, with respect to increasing antigen concentration (FIG. 16A) and antiserum dilution (FIG.16B), as depicted on x-axis. The antigen binding for antiserum from each mouse within the Hu CDKL5a group on day 63 post-immunization (denoted as Hu CDKL5a m1, D63, Hu CDKL5a m2, D63, and Hu CDKL5a m3, D63), Hu CDKL5s group on day 63 post-immunization (denoted as Hu CDKL5s m1, D63, Hu CDKL5s m2, D63, and Hu CDKL5s m3, D63), one mouse from Hu CDKL5s group on an earlier time point control, day 35, post-immunization (denoted as Hu CDKL5s m1, D35), and pre-immune serum, are as indicated.

[0129] FIGs.17A-17C are ELISA plots showing target antigen binding by antibody in media isolated from hybridoma tissue culture prepared from tagged CDKL5 antigen immunized mice. FIGs. 17A-17B depict antigen binding by hybridoma culture media from tagged-CDKL5-Hu CDKL5s immunization group. The absorbance showing level of binding to untagged CDKL5 protein is depicted on the y-axis with respect to increasing antigen concentration (FIG. 17A), and hybridoma tissue culture (HT) media or antiserum dilution (FIG. 17B), as depicted on the x-axis. The antigen binding for the hybridoma tissue culturemedia (Hu CDKL5a-2HL media), the corresponding antiserum pooled from mice 1 and 3 of the Hu CDKL5a group isolated at day 35 (labelled as Hu CDKL5a-2 m1 & m3, D35 FS) , and HT media (negative control), are as indicated. FIG.17C depicts antigen binding by hybridoma culture media from tagged-CDKL5-Hu CDKL5a immunization group. The absorbance showing level of binding to untagged CDKL5 protein is depicted on the y-axis with respect to increasing HT media or antiserum dilution, as depicted on the x-axis. The antigen binding for the hybridoma tissue culture media (Hu CDKL5a HT media), the corresponding antiserum from the mouse of the Hu CDKL5a group isolated at day 35, and HT media (negative control), are as indicated.

[0130] FIGs.18A-18B are western blots showing antigen binding of antibody purified from media of twelve monoclonal antibody producing clones (denoted as 2M1, M2, 2M3, M3, 2M4, 2M6, 2M7, 2M9, 2M11, 2M12, 2M13, and 2M14) and two commercially available CDKL5 antibodies (denoted as “Invitrogen MA531406” and “Santa Cruz SC376314”). The molecular weight marker (“M”), SPY- μDys (negative control, ”-”),1 μg SPYcoupled-CDKL5 (“S”), and 1 μg CDKL5-kinase (“K”), were run on a 4-20% TGX gel, as indicated, and transferred to PVDF membrane followed by primary probing with each antibody isolated from CDKL5 monoclonal antibody producing clone (FIG.18A) or the two commercially available antibodies) (FIG. 18B). Primary antibody concentration was 0.4 μg / ml, and secondary rabbit anti-mouse IgG H&L chain antibody concentration was 0.664 μg / ml. The bands indicating binding to CDKL5 antigen are indicated with arrows.

[0131] FIG.19 is an ELISA plot showing antigen sensitivity of antibody isolated from media of twelve monoclonal antibody producing clones (denoted as 2M1, M2, 2M3, M3, 2M4, 2M6, 2M7, 2M9, 2M11, 2M12, 2M13, and 2M14). The optical density (450nm) at y-axis shows the level of CDKL5 binding corresponding to increasing concentration (x-axis) of the twelve monoclonal CDKL5 specific antibodies of the present disclosure, as indicated.

[0132] FIGs. 20A-20L are HPLC-SEC analysis plots from stability test of twelve CDKL5 specific monoclonal antibodies (2M1, M2, 2M3, M3, 2M4, 2M6, 2M7, 2M9, 2M11, 2M12, 2M13, and 2M14, respectively) after freeze and thaw cycles. The analysis plots of each monoclonal antibody in non-frozen state (denoted as “Not frozen”) and after a first and second freeze / thaw cycle (F / T cycle 1 and F / T cycle 2), are as indicated on each plot.

[0133] FIGs. 21A-21C are SDS-PAGE gel images showing stability and integrity of twelve CDKL5 specific monoclonal antibodies (denoted as 2M1, M2, 2M3, M3, 2M4, 2M6, 2M7, 2M9, 2M11, 2M12, 2M13, and 2M14). FIGs.21A and 21B are SDS-PAGE images of twelve CDKL5 specific monoclonal antibodies in non-frozen state and after a first and second freeze / thaw cycle (F / T 1 and F / T 2) run on a 4-20% TGX gel, as indicated. The 150 kDa band of full length antibody (“intact antibody” in FIG.21A and “intact IgG” in FIG.21B) is indicated by an arrow. The lanes used to run the marker (M), non-frozen antibody (NF), freeze / thaw cycle 1 (F / T1) and freeze / thaw cycle 2(F / T2) are as indicated. FIG.21C is a SDS-PAGE image of twelve CDKL5 specific monoclonal antibodies run on a 4-20% TGX gel under reducing (R) and non-reducing (NR) conditions, as indicated. The full length intact antibody, and the heavy and light chains of the reduced antibody are as indicated.

[0134] FIGs. 22A-22B are immunocytochemistry images showing specific detection of human CDKL5 in a mouse neuronal cell line. FIG. 22A depicts immunocytochemistry images of wild type Neuro 2A cells (WT), CDKL5 knockout Neuro 2A cells (CDKL5 KO) cells, and Neuro 2a CDKL5 KO cells transfected with human CDKL5 encoding plasmid (CDKL5 KO + hCDKL5) stained with three anti-CDKL5 monoclonal antibodies (M2, M3 and 2-M9 (“2M9”)) of the present disclosure, as indicated. Representative images show detection of hCDKL5 protein only in transfected cells using the three anti-CDKL5 antibodies, 2M9, M2 (IgG1) and M3 (IgG1). Scale bar 50µm. FIG. 22B depicts images of in situ hybridization results using BaseScopeTMstaining of WT, CDKL5 KO, and CDKL5 KO + hCDKL5 mouse Neuro 2A cells, using a probe against hCDKL5 mRNA, as indicated.

[0135] FIG. 23 is a chart depicting results from CDKL5 binding by antibody pairs using the Octet Red96e-SAX biosensor assay. The solid cells indicate antibody pairs that block each other (cross-blocking) from binding to CDKL5 possibly due to binding of same or overlapping epitopes. The blank cells indicate antibody pairs that do not block each other (non- blocking) from binding to CDKL5 thus indicating binding to distinct or non-overlapping epitopes of CDKL5. The crossed cells indicate self-blocking in wells containing the same anti- CDKL5 antibody as both members of the antibody pair. Two distinct sets of cross-blocking antibodies, Bin1 and Bin 2, are indicated.

[0136] FIGs.24A-24E shows HPLC-SEC analysis plots of Blank (buffer only) (FIG. 24A), full length CDKL5 (FIG.24B), and various standards (IgM (FIG.24C), thyroglobulin(FIG. 24D) and ferritin (FIG. 24E)), as indicated, run using a wide-pore AdvanceBio SEC column. DETAILED DESCRIPTION

[0137] All publications, patents, and patent applications, including any drawings and appendices therein, are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application, drawing, or appendix was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0138] Throughout the description, where compositions (e.g., proteins) are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions (e.g., proteins) of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0139] In the present specification, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0140] Throughout the present specification, numerical ranges are provided for certain quantities. It is to be understood that these ranges comprise endpoints and all subranges therein, including each integer in and between a disclosed range. Thus, the range “from 50 to 80” includes all possible ranges therein (e.g., 51-79, 52-78, 53-77, 54-76, 55-75, 60-70, etc.) as well as each individual integer from 50 to 80 (e.g., 50, 51, 52, 53, 54, etc.). Where ranges are provided in the form or fractions, percentages, decimals, and the like, such ranges likewise include all possible subranges therein and each individual fraction, percentage, decimal, etc. in and between the disclosed range. For example, the range “from 0.1 to 1.0” includes all possible ranges therein (e.g., 0.2 to 0.9, etc.) and each individual 1 / 10thdecimal from 0.1 to 1.0 (e.g., 0.1, 0.2, 0.3, 0.4, etc.). Furthermore, all values within a given range may be an endpoint for therange encompassed thereby (e.g., the range 50-80 includes the ranges with endpoints such as 55-80, 50-75, etc.).

[0141] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the presently described methods remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0142] The use of any and all examples, or exemplary language herein, for example, “such as” or “including” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.

[0143] Further, it should be understood that elements and / or features of a composition (e.g., proteins) or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions (e.g., proteins) of the present invention and / or in methods of the present invention, unless otherwise understood from the context. In other words, within the present disclosure, embodiments have been described and depicted in a way that enables a clear and concise disclosure to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein. Definitions

[0144] The following terms used in the present specification and claims are believed to be well understood by one of ordinary skill in the art given their use and context herein. The following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0145] The term “a” or “an” refers to one or more of that entity; for example, “a CDKL5 protein” refers to one or more CDKL5 proteins or at least one CDKL5 protein unless context dictates otherwise. As such, the terms “a” (or “an”), “one or more” and “at least one” are used interchangeably herein unless context dictates otherwise. In addition, reference to, e.g., “anagent” by the indefinite article “a” or “an” does not exclude the possibility that more than one of agents is present, unless the context clearly requires that there is one and only one agent present.

[0146] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ± 10% variation from the nominal value unless otherwise indicated or inferred.

[0147] It should be understood that the expression “at least one of” includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and / or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0148] The term “affinity tag” refers to a peptide domain that can be added to a recombinantly expressed protein (i.e., “recombinant protein”) to aid in the separation, isolation, and / or purification of the recombinant protein. Typically, an affinity tag comprises an epitope or interaction domain that can be specifically recognized by a known antibody or that interacts with high affinity to a known reagent or material. A chromatography resin or other solid substrate can then be prepared with the antibody or reagent / material immobilized thereon, creating a substrate for the separating, isolating, and / or purifying of the recombinant protein comprising the affinity tag. Examples of affinity tags suitable for use in the recombinant proteins and methods disclosed herein include but are not limited to polyhistidine (e.g., 6xHIS (SEQ ID NO: 21)) tags, S-tag, glutathione-S-transferase (GST), streptavidin and variants thereof, protein A, FLAG peptides, green fluorescent protein (GFP), and maltose-binding protein (MBP) tags.

[0149] The term “cleavage site linker” refers to a peptide domain or a chemical linker between domains of a recombinantly expressed protein that can be specifically cleaved by defined conditions, chemical, or enzymatic cleavage. Such cleavage sites enable removal of, e.g., affinity tags or solubility tags or other domains fused to the recombinant proteins as expressed. Examples of protein cleavage sites suitable for use in the recombinant proteins and methods disclosed herein include but are not limited to peptide cleavage sites, disulfidecleavage sites, peptide sites recognized by intracellular proteases (lysosomal cleavage sites), and Tobacco etch virus (TEV) protease cleavage site.

[0150] The term “detection tag” refers to a peptide domain that can be added to a recombinantly expressed protein (i.e., recombinant proteins) to aid in the detection of the recombinant protein, e.g., recombinant protein expressed in a cell or tissue or present in sample. Detection tags include fluorescence-based and immunoaffinity based tags which can be detected in a tissue or a sample using, e.g., light or fluorescence microscopy and / or immunoaffinity-based cross-linking and detection. In some instances, detection tags can be used as affinity tags, and vice versa.

[0151] The term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0152] The term “lysis buffer” refers to a solution suitable for use in rupturing or lysing cells or disrupting or disintegrating cell membranes to facilitate separation of cytosolic and membrane-based fractions of the cells thus enabling extracting proteins from cytoplasmic, nucleus or membrane fractions of lysed cells or a combination thereof. For example, a lysis buffer may be used to disrupt (lyse, break, tear, or destabilize) cellular membranes of cells present in a cell culture, enabling separation of a cytosolic fraction (supernatant) and membrane fraction (pellet) by centrifugation of the lysed cells. As described further herein, lysis buffers may comprise a detergent, e.g., a cationic detergent, an anionic detergent, or a zwitterionic detergent.

[0153] The term “non-denatured” as it relates to proteins refers to the 3-dimensional structure and conformation of a protein as it exists in a cellular environment, cellular milieux, and / or extracellular environment / milieux. That is, non-denatured refers to a protein as it exists in its physiological state, including secondary, tertiary, and quaternary folding structure and binding / interaction partners. Often, denaturation steps are implemented to detect or isolate expressed proteins. Such denaturation steps may include cleavage of disulfide bonds, covalent cross-linking, stripping of glycosylation and other post-translational modifications, and / or disruption of non-covalent interactions such as intra-protein interactions between protein domains and / or interactions between proteins (such homomultimeric and / or heteromultimericinteractions). Thus, non-denatured refers to a protein that retains these structural, conformational, and intra- and inter-protein interaction characteristics. Non-denatured proteins may retain some or all of the structural, conformational, and intra- and inter-protein interaction characteristics of the expressed protein as it exists in a cellular environment, cellular milieux, and / or extracellular environment / milieux.

[0154] The term “recombinant human CDKL5” (rhCDKL5) protein comprises engineered, truncated, and otherwise modified variants of a wild type or native CDKL5 protein. The rhCDKL5 proteins (referred to at times throughout the specification simply as rhCDKL5s) are non-naturally occurring recombinant polypeptides comprising specific peptide tags to a CDKL5 polypeptide that enable their soluble expression while retaining advantageous and / or important biological functions of a wild type or native CDKL5 protein. Generally speaking, the wild type or native CDKL5 protein refers to the human polypeptide sequence of a brain isoform of CDKL5 (CDKL5 isoform 2) consisting of 960 amino acid residues (SEQ ID NO: 1, canonical human CDKL5 protein sequence, UniProt ID no. O76039, NCBI RefSeq no. NP_001032420) or an isoform of CDKL5 (CDKL5 isoform 1) consisting of 1030 amino acid residues (SEQ ID NO: 2, NCBI RefSeq no. NP_001032420.1). The kinase / catalytic domain of CDKL5 protein comprises the portion of CDKL5 from amino acid positions 1 to 287. The non- catalytic / non-kinase domain also referred to as the disordered domain, the C-terminal regulatory domain or COOH-domain comprises the portion of CDKL5 from amino acid position 288 to the last C-terminal amino acid. The disordered domain also comprises two nuclear localization signal sequences, NLS1 and NLS2.

[0155] The term “soluble” or “solubilized” or “released” and the like, as used in the context of expressed proteins, refers to the status of the protein in a liquid buffer to form a solution with that buffer. If the protein is soluble, it is in solution or it can be solubilized into solution or released from interactions with other proteins / components to be in solution in that buffer. Cells can be separated into cytosolic and membrane fractions. Proteins expressed in cells (intracellular proteins) may be associated with the cytosol or may be associated with a membrane. Proteins that are membrane-endogenous, such as transmembrane proteins and other proteins that are fully or partially embedded in membranes or otherwise strongly interact with membranes, may be difficult to solubilize for purification. The process of extracting such proteins from membranes is referred to as solubilization. A protein so extracted may be referred to as “released” from the membrane.

[0156] The terms “intracellular expression tag” or “solubility tag” are used interchangeably and refer to a domain that can be added to recombinantly expressed proteins (i.e., recombinant proteins) and that serve to increase the solubility of the recombinant protein. Without wishing to be bound by theory, intracellular expression tags may function to destabilize protein interactions by promoting proper folding, increasing translation initiation of the recombinant protein, and / or providing polypeptide chains rich in positively charged amino acids that increase electrostatic repulsion during translation reducing / avoiding aggregation. Examples of intracellular expression tags suitable for use in the recombinant proteins and methods described herein include maltose-binding protein tag (MBP tag) and green fluorescent protein (GFP). In some instances, intracellular expression tags may also function as affinity tags.

[0157] As used herein, unless otherwise indicated, the term “antibody” is understood to mean an intact antibody (e.g., an intact monoclonal antibody), or a fragment thereof, such as a Fc fragment of an antibody (e.g., an Fc fragment of a monoclonal antibody), or an antigen- binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody), including an intact antibody, antigen-binding fragment, or Fc fragment that has been modified, engineered, or chemically conjugated. Examples of antigen-binding fragments include Fab, Fab’, (Fab’)2, Fv, single chain antibodies (e.g., scFv), minibodies, and diabodies.

[0158] The terms “CDKL5 specific monoclonal antibody”, “CDKL5 specific antibody”, “anti-CDKL5 monoclonal antibody”, “anti-CDKL5 antibody”, “CDKL5 mAb”, and the like, as used herein refer to one or more monoclonal antibodies with specificity for CDKL5 protein; i.e., one or more anti-CDKL5 antibody / ies produced from a cell lineage made by cloning a unique single cell derived from a hybridoma culture. All antibodies produced by this single cell clone have the same epitope specificity and isotype.

[0159] The term “freeze / thaw”, “F / T” or “F / T cycle”, as used herein is a process of subjecting a sample of a biological or chemical substance, like a CDKL5 specific monoclonal antibody, to temperature of less than or equal to freezing for a period of time followed by subjecting the same sample to room temperature for a period of time. In some embodiments, the sample may be subjected to a temperature less than or equal to -20℃ for 10 minutes to 1 hour, or more, then subjected to room temperature for 10 minutes to 1 hour, or more.

[0160] The term “reducing condition”, as used herein is a process of denaturing or breaking the intra-chain disulfide bonds within the heavy and light chains of a monoclonal antibody and interchain di-sulfide bonds between the two heavy chains and between a pair of heavy and light chains of the antibody, by contacting the antibody with a suitable reducing agent, for example, tris (2-carboxyethyl) phosphine hydrochloride (TCEP), beta- mercaptoethanol (BME), or dithiothreitol (DTT).

[0161] The term “association rate constant” (Ka, Kon or on-rate) as used herein is the rate at which one molecule (e.g., an antibody) binds to a second molecule (e.g., an antigen) to form a complex. It is recorded in moles (the standard quantification of the number of molecules) per second: M-1s-1. The association rate constant is directly proportional to the strength / affinity of binding of the first molecule with the second molecule.

[0162] The term ‘dissociation rate constant” (Kd, Koff or off-rate) as used herein is a measurement of the propensity at which one molecule (e.g., an antibody) separates or dissociates from a complex with a second molecule (e.g., an antigen). The dissociation rate constant is indirectly proportional to the strength / affinity of binding of the first molecule with the second molecule.

[0163] The term “equilibrium constant” or “equilibrium dissociation constant” (KD) refers to the equilibrium constant of the dissociation, calculated by dividing the off-rate Kd by the on-rate Ka.

[0164] The term “first order rate constant” (Kobs) as used herein refers to the constant proportion of a molecule (e.g., an antibody) that is eliminated from system (e.g., an in vitro binding reaction or a body) per unit of time in a first-order exponential rate.

[0165] The term “Rmax”, as used herein refers to the maximal feasible surface plasmon resonance (SPR) signal generated by an interaction between a ligand – analyte pair (e.g., an antibody and an antigen pair) and is represented in response units (RU).

[0166] The term response “Req” refers to steady state equilibrium of a ligand–analyte pair binding reaction (e.g., an antibody and an antigen pair binding reaction), and is dependent on the number of ligand binding sites, the analyte concentration (C) and the equilibrium dissociation constant (KD). The Req can be calculated with the following equationIntroduction

[0167] The present disclosure provides a reliable method for producing soluble, non- denatured and full-length recombinant human CDKL5 (rhCDKL5) protein according to various human CDKL5 protein sequences for treating CDKL5 deficiency disorder (CDD) (See, for example, Accession Nos: NP_001310218.1, NP_001032420.1, NP_003150.1, EAW98941.1 and ADN38258.1). The recombinant CDKL5 therapeutic proteins based on the sequences disclosed herein are typically delivered to central nervous system using viral gene therapy vectors comprising a vector genome encoding the recombinant human CDKL5 protein. For example, multiple candidate AAV gene therapies are being evaluated for their ability to deliver functional recombinant human CDKL5 protein to brain tissue of patients suffering from CDD.

[0168] A critical aspect of demonstrating efficacy of such therapies is showing that administration of the gene therapy vectors to a subject result in meaningful expression of the rhCDKL5 protein in neuronal tissue of the subject. The obverse is also true. It is important to show what level of expression is required in neuronal tissue to establish efficacy in a subject. Further, the rhCDKL5 should be localized to both the cell membrane and nucleus of neuronal cells and should interact and phosphorylate proper target protein partners of CDKL5 involved in neuronal development and functioning, i.e., centrosomal protein 131 (CEP131), microtubule-associated protein RP / EB family member 2 (MAP1S / EB2), Rho-Rac guanine nucleotide exchange factor 2 (ARHGEF2), Disks large homolog 5 (DLG5), Shootin 1 (SHTN1), Cytoplasmic linker protein 170 (CLIP170), IQ Motif Containing GTPase Activating Protein 1 (IQGAP1), Elongin A (ELOA), Trichothiodystrophy non-photosensitive 1 (TDN1), E1a binding protein P400 (EP400), Methyl-CpG binding protein 2 (MeCP2), DNA- methyltransferase 1 (DNMT1), Histone deacetylase 4 (HDAC4), Transcription factor SOX-9 (SOX9), Mothers against decapentaplegic homolog 3 (SMAD3), Amphiphysin 1 (AMPH1) and Netrin-G ligand-1 (NGL1). Further, it will be important to demonstrate the levels of such proteins present in neuronal tissue of subjects administered rhCDKL5-based therapeutics, including gene therapies. Accordingly, quantitative methods of measuring rhCDKL5 protein present in subject samples are needed.

[0169] Previous attempts to develop such methods have been hampered by the difficulty in reliably expressing and purifying rhCDKL5 protein in vitro because a source of such rhCDKL5 protein is needed to calibrate quantitative assays. The presently disclosedmethods thus enable quantitative measurement of rhCDKL5 protein present in samples, e.g., from subject tissue samples (e.g. cerebrospinal fluid (CSF)), by providing a source of recombinant protein that can be used as a comparator and to generate a calibration curve in quantitative assays.

[0170] The presently disclosed methods also enable further in vitro characterization of the rhCDKL5 proteins to better understand their function, their intracellular trafficking, and their binding / interaction partners.

[0171] Another critical aspect of evaluating efficacy and safety of rhCDKL5-based therapeutics, including viral vector-mediated rhCDKL5s, is the ability to detect antibodies in subjects that react with the rhCDKL5 proteins. Immune responses in some subjects that are administered therapeutic drugs, including proteins and therapeutic transgenes, are known to give rise to anti-drug antibodies (ADAs). This immune response can curtail the efficacy of a therapy over time in the subject and can also reduce the efficacy of the same therapy upon re- administration. Accordingly, methods for detecting ADAs in subjects administered rhCDKL5- based therapeutics as well as in subjects to be administered such therapeutics are needed. However, the lack of reliable methods for producing full-length, soluble and non-denatured rhCDKL5 protein has made it difficult to develop these methods. Thus, the presently disclosed methods also enable assays for detecting anti-CDKL5 antibodies in samples from subjects.

[0172] The presently disclosed methods achieve these and other ends by providing specifically tagged rhCDKL5 proteins and assays for producing and purifying the tagged and tagless CDKL5 proteins that result in reliable expression of recombinant human CDKL5 proteins that are soluble and non-denatured. Recombinant Human CDKL5 (rhCDKL5) Proteins

[0173] Wild type human CDKL5 protein is 960 amino acids in length and approximately 107.5 kDa in size (SEQ ID NO: 1, UniProt ID no. O76039, NCBI RefSeq no. NP_001032420 encoded by human CDKL5 gene, NCBI RefSeq no. NM_001037343.2) or 1030 amino acids in length and approximately 115.6 kDa in size (SEQ ID NO: 2, NCBI RefSeq no. NP_001032420.1). The protein is organized into several structural and functional domains, as shown in FIG.1A, including: an N-terminal catalytic domain (residues 1-287); a C-terminal regulatory domain (residues 288-938) and N-terminal nuclear localization signal 1 (NLS-1, residues 312-315, KRKP (SEQ ID NO: 112)) and N-terminal nuclear localization signal 2(NLS2, residues 784-789, KKKKKK (SEQ ID NO: 3). The CDKL5 protein comprises several intrinsically disordered protein (IDP) domains / structures within two-thirds of the length of the protein mainly in the regulatory domain. The N-terminal kinase / catalytic domain of CDKL5 protein comprises the portion of CDKL5 from amino acid positions 1 to 287. The C-terminal regulatory domain also referred to as the non-catalytic / non-kinase domain, the disordered domain, or COOH-domain comprises the portion of CDKL5 from amino acid position 288 to the last C-terminal amino acid. The disordered domain also comprises two nuclear localization signal sequences, NLS1 and NLS2.

[0174] An Intrinsically Disordered Protein (IDP) lacks a fixed or ordered three- dimensional structure. It has been proposed that the flexibility of disordered proteins facilitate different conformation requirements for binding modifying enzymes and receptors. IDPs like CDKL5 adapt to many different structures in vivo according to the cell’s conditions, creating a structural or conformational ensemble. The existence and kind of protein disorder is encoded in its amino acid sequence. IDPs generally have a low content of bulky hydrophobic amino acids and a high proportion of polar and charged amino acids. This property leads to good interactions with water. CDKL5 is an intrinsically disordered protein that, when produced recombinantly, is either subject to a massive proteolytic attack by host-encoded proteases or tends to form aggregates. Although, the functionally active protein kinase domain has been successfully expressed and purified from E. coli, there is only one example of expression of soluble full-length recombinant protein of hCDKL5, in an Antarctic bacterium at subzero temperatures. There are no known examples of successfully making and purifying the active full-length protein in a mammalian cell expression system.

[0175] Particularly, it is not known how to stabilize and protect the full-length human CDKL5 protein in solution from aggregation and proteolytic attack. The present disclosure provides methods to express and purify full-length CDKL5 with and without tags from a mammalian cell expression system. The portion of the tagged full-length CDKL5 proteins disclosed herein comprise of an N-terminal catalytic domain and a C-terminal regulatory domain (comprising a nuclear localization signal 1 domain and a nuclear localization signal 2 domain) and is referred to as the CDKL5 sequence. The methods disclosed herein provide full- length human CDKL5 proteins with purity greater than 90%, enabling delivery in a monomeric state suitable for injection in subjects for therapeutic application and antibody production. The full-length human CDKL5 proteins generated using the methods of the present disclosure canbe delivered in a formulation that stabilizes the proteins for labeling. The portion of the rhCDKL5 proteins disclosed herein comprising an N-terminal catalytic domain and a C- terminal regulatory domain, wherein the C-terminal regulatory domain comprises a nuclear localization signal 1 domain and a nuclear localization signal 2 domain, and is referred to as the CDKL5 sequence.

[0176] In certain embodiments, the CDKL5 sequence comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the CDKL5 sequence of the rhCDKL5 comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2. rhCDKL5 with intracellular expression tag

[0177] The rhCDKL5 proteins produced by the methods described herein comprise a CDKL5 sequence comprising an N-terminal catalytic domain and a C-terminal regulatory domain comprising a nuclear localization signal 1 domain and a nuclear localization signal 2 domain, and one or more intracellular expression tags. In certain embodiments, the CDKL5 sequence comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the CDKL5 sequence of the rhCDKL5 comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0178] The rhCDKL5 protein produced by the methods described herein, can comprise a green fluorescent protein (GFP) tag comprising an amino acid sequence of any of the GFP proteins known in the art, including but not limited to amino acid sequences according to NCBI RefSeq Nos. CAB65651.1, ACI46135.1, ACI46133.1, AAN86137.1, BAM36532.1, BAG71039.1, WGG63471.1 and P42212.1. In certain embodiments, the rhCDKL5 protein produced by the methods described herein, can comprise a GFP tag comprising an amino acid sequence according to SEQ ID NO: 39. The rhCDKL5 protein produced by the methods described herein, can comprise a maltose binding protein (MBP) tag comprising an amino acid sequence of any of the MBP proteins known in the art, including but not limited to amino acid sequences according to NCBI RefSeq Nos. AAN86140.1, AAN86139.1, AAN86138.1,AAN86137.1, BAM71967.1, AEJ45096.1, ADX83587.1, ADX86244.1, ADC95619.1, AAC83813.1, ABO28850.1, EEP73237.1, ADO71329.1, QSV51880.1 and KEZ16092.1. In certain embodiments, the rhCDKL5 protein produced by the methods described herein, can comprise a MBP tag comprising an amino acid sequence according to SEQ ID NO: 38. In certain embodiments, the rhCDKL5 protein produced by the methods described herein, can comprise a combination of a GFP tag and an MBP tag, as disclosed herein.

[0179] The rhCDKL5 protein produced by the methods described herein, can be linked to at least one intracellular expression tag by a Tobacco etch virus (TEV) protease cleavage site linker. In certain embodiments, the TEV protease cleavage site linker comprises ENLYFQS (SEQ ID NO: 19) or ENLYFQG (SEQ ID NO: 20). In some embodiments, the rhCDKL5 protein is linked to an MBP tag at the N-terminus by a TEV protease cleavage site linker and linked to a GFP tag at the C-terminus by a TEV protease cleavage site linker. In some embodiments, the rhCDKL5 protein linked to an MBP tag at the N-terminus by a TEV protease cleavage site linker and linked to a GFP tag at the C-terminus by a TEV protease cleavage site linker comprises an amino acid sequence that shares at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the rhCDKL5 protein linked to an MBP tag at the N-terminus by a TEV protease cleavage site linker and linked to a GFP tag at the C-terminus by a TEV protease cleavage site linker comprises an amino acid sequence set forth in SEQ ID NO: 4.

[0180] In some embodiments, the rhCDKL5 protein is linked to a GFP tag at the N- terminus by a TEV protease cleavage site linker and linked to an MBP tag at the C-terminus by a TEV protease cleavage site linker. In some embodiments, the rhCDKL5 protein linked to a GFP tag at the N-terminus by a TEV protease cleavage site linker and linked to an MBP tag at the C-terminus by a TEV protease cleavage site linker comprises an amino acid sequence that shares at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the rhCDKL5 protein linked to a GFP tag at the N-terminus by a TEV protease cleavage site linker and linked to an MBP tag at the C-terminus by a TEV protease cleavage site linker comprises an amino acid sequence set forth in SEQ ID NO: 5.

[0181] In some embodiments, the rhCDKL5 protein comprises a biotinylable peptide tag, GLNDIFEAQKIEWHE (SEQ ID NO: 14) linked to the N- or C-terminus of any of the rhCDKL5 proteins disclosed herein. In certain embodiments, the biotinylable peptide tag is located between an intracellular expression tag and the C-terminus of the rhCDKL5 protein. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence according to the SEQ ID NO: 18. In certain embodiments, the rhCDKL5 protein can be a CDKL5 partner 1 protein, wherein the rhCDKL5 protein comprises an MBP-TEV tag at the C-terminus of the rhCDKL5 protein and a GFP-TEV tag at the N-terminus of the rhCDKL5 protein. The rhCDKL5 proteins produced by the methods of the present disclosure, can be biotinylated by linking the biotinylable peptide tag (e.g., biotinylable peptide tag of SEQ ID NO: 14) of the rhCDKL5 protein to a biotin compound. The linking of the biotinylable peptide tag (e.g., biotinylable peptide tag of SEQ ID NO: 14) of the rhCDKL5 protein to a biotin compound can be done in cells of host cell culture that express a biotin ligase enzyme selected from any one of the biotin ligases BirA, BiolD, TurboID, BiolD2, Air ID, MicroID2 and Apex. The biotin ligase enzyme can be E. coli biotin ligase, BirA.

[0182] In some embodiments, the rhCDKL5 protein comprises a CDKL5 that comprises a peptide tag (e.g., a peptide tag comprising an amino acid sequence of any one of SEQ ID NOs: 30-32 or a fragment thereof, (e.g., partner 1 peptide tag) linked to the N- or the C-terminus of any of the rhCDKL5 proteins disclosed herein to generate a CDKL5 partner 1 protein. In certain embodiments, the CDKL5 partner 1 protein can bind (e.g., irreversibly or reversibly) to a partner 2 protein comprising an amino acid sequence according to any one of SEQ ID NOs: 33-35 or a fragment thereof. In certain embodiments, the partner 2 protein is labeled with a detectable moiety. The partner 1 protein and the partner 2 protein can be labeling and conjugating partner 1 peptide / partner 2 protein systems known in the art, including but not limited to BioRad SpyCatcher and SpyTag products, Kerafast Biotech SpyTag / SpyCatcher Protein Coupling Reagents or Addgene SpyTag-β-Lactamase-SpyCatcher proteins. In certain embodiments, the partner 1 peptide tag or a fragment thereof can be linked to the N-terminus of a rhCDKL5 protein comprising an amino acid sequence according to SEQ ID NO: 5. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the rhCDKL5 protein (e.g., a rhCDKL5 protein linked toa partner 1 peptide tag at the N-terminus) can comprise an amino acid sequence according to SEQ ID NO: 22. In certain embodiments, the partner 1 peptide tag or a fragment thereof can be located between the GFP-TEV linker and the N-terminus of the CDKL5 sequence in the rhCDKL5 protein comprising an amino acid sequence according to SEQ ID NO: 5. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 23.

[0183] In certain embodiments, the partner 1 peptide tag or a fragment thereof can be linked to the C-terminus of a rhCDKL5 protein comprising an amino acid sequence according to SEQ ID NO: 5. In certain embodiments, the rhCDKL5 protein linked to a partner 1 peptide tag at the C-terminus can comprise an amino acid sequence according to SEQ ID NO: 24. In certain embodiments, the partner 1 peptide tag or a fragment thereof can be located between the C-terminus of the CDKL5 sequence and the MBP-TEV linker in the rhCDKL5 protein comprising an amino acid sequence according to SEQ ID NO: 5. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 25. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 25. rhCDKL5 with secretion signal tag

[0184] The rhCDKL5 proteins produced by the methods described herein comprise a CDKL5 sequence comprising an N-terminal catalytic domain and a C-terminal regulatory domain comprising a nuclear localization signal 1 domain and a nuclear localization signal 2 domain, and one or more secretion signal tags. In certain embodiments, the CDKL5 sequence may comprise an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the CDKL5 sequence may comprise an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ IDNO: 1. In some embodiments, the CDKL5 sequence of the rhCDKL5 comprising one or more secretion signal tags produced by the methods described herein comprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, CDKL5 sequence may comprise an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0185] In the rhCDKL5 proteins comprising one or more secretion tags produced by the methods disclosed herein, the secretion tag can be a kappa light chain signal sequence tag. In certain embodiments, the kappa light chain signal sequence tag comprises an amino acid sequence MDMRVPAQLLGLLLLWLPGAKC (SEQ ID NO: 6). In certain embodiments, the kappa light chain signal sequence tag comprising SEQ ID NO: 6 is linked to the N-terminus and / or the C-terminus of the rhCDKL5 protein. In certain embodiments, the secretion signal tag can be a heavy chain signal sequence tag. In certain embodiments, the heavy chain signal sequence tag comprises an amino acid sequence MGVKVLFALICIAVAEA (SEQ ID NO: 7). In certain embodiments, the heavy chain signal sequence tag comprising SEQ ID NO: 7 is linked to the N-terminus and / or the C-terminus of the rhCDKL5 protein. In certain embodiments, the secretion signal tag can be a hemagglutinin signal sequence tag. In certain embodiments, the hemagglutinin signal sequence tag comprises an amino acid sequence MKTIIALSYIFCLVFA (SEQ ID NO: 8). In certain embodiments, the hemagglutinin signal sequence tag is linked to the N-terminus and / or the C-terminus of the rhCDKL5 protein.

[0186] In certain embodiments, the rhCDKL5 proteins comprising one or more secretion tags produced by the methods disclosed herein comprises an affinity purification tag . In certain embodiments, the affinity purification tag is linked to the N-terminus of the rhCDKL5 protein when the rhCDKL5 protein has at least one secretion signal tag linked to the C-terminus of the rhCDKL5 protein. In certain embodiments, the affinity purification tag is linked to the C-terminus of the rhCDKL5 protein when the rhCDKL5 protein has at least one secretion signal tag linked to the N-terminus of the rhCDKL5 protein. In certain embodiments, the affinity purification tag is selected from: a polyhistidine tag, a polyarginine tag, a glutathione-S-transferase tag, a DYKDDDDK (SEQ ID NO: 29) peptide tag, a streptavidin- binding peptide tag, a peptide tag comprising an amino acid sequence according to any one of SEQ ID NOs: 36 or 37, a calmodulin binding peptide tag, a maltose-binding tag, and a cellulose-binding tag. In certain embodiments, the polyhistidine tag is a 6xHIS tag (SEQ ID NO: 21).

[0187] In certain embodiments, the rhCDKL5 proteins produced by the methods disclosed herein comprise a kappa light chain signal sequence tag linked to the N-terminus of the rhCDKL5 protein and a 6xHIS tag (SEQ ID NO: 21) linked to the C-terminus of the rhCDKL5 protein. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 9. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 9.

[0188] In certain embodiments, the rhCDKL5 proteins produced by the methods disclosed herein comprise a heavy chain signal sequence tag linked to the N-terminus of the rhCDKL5 protein and a 6xHIS tag (SEQ ID NO: 21) linked to the C-terminus of the rhCDKL5 protein. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 10.

[0189] In certain embodiments, the rhCDKL5 proteins produced by the methods disclosed herein comprise a hemagglutinin signal sequence tag linked to the N-terminus of the rhCDKL5 protein and a 6xHIS tag (SEQ ID NO: 21) linked to the C-terminus of the rhCDKL5 protein. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 11. rhCDKL5 with exosome trafficking tag

[0190] The rhCDKL5 proteins produced by the methods described herein comprise a CDKL5 sequence having an N-terminal catalytic domain and a C-terminal regulatory domain comprising a nuclear localization signal 1 domain and a nuclear localization signal 2 domain, and an exosome trafficking tag. In certain embodiments, the CDKL5 sequence may comprise an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical tothe amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the CDKL5 sequence may comprise an amino acid sequence that is at least at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the CDKL5 sequence of the rhCDKL5 comprising an exosome trafficking tag produced by the methods described herein comprise an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the CDKL5 sequence of the rhCDKL5 may comprise an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 2.

[0191] In some embodiments, the rhCDKL5 proteins produced by the methods described herein comprise an exosome trafficking tag. In certain embodiments, the exosome trafficking tag is an exosome-targeting viral peptide, or a fragment thereof. In certain embodiments, the exosome trafficking tag is linked to the N-terminus or the C-terminus of the rhCDKL5 protein. In certain embodiments, the exosome trafficking tag comprises an HIV GAG protein or a fragment thereof, an HIV NEF peptide, an HTLV-1 transactivator protein (Tax), an HCV core protein, or an HCV glycoprotein E2. In certain embodiments, the exosome trafficking tag is an HIV GAG protein or a fragment thereof. In certain embodiments, the HIV GAG protein or fragment thereof is linked at the N- or C-terminus of the rhCDKL5 protein. In certain embodiments, the HIV GAG protein or a fragment thereof is linked to the N-terminus of the rhCDKL5 protein. In some embodiments, the HIV GAG protein or fragment thereof linked to the rhCDKL5 protein can be derived from a wild type HIV GAG protein comprising amino acid mutation selected from L31R, V35E, W36A or a combination thereof. In some embodiments, the HIV GAG protein or fragment thereof linked to the rhCDKL5 protein can be derived from a wild-type HIV GAG protein comprising amino acid mutations L31R, V35E and W36A. In certain embodiments, the HIV GAG protein comprises an amino acid sequence according to SEQ ID NO: 12. In certain embodiments, the rhCDKL5 protein comprising the exosome trafficking tag comprises an affinity purification tag. In certain embodiments, the affinity purification tag is linked to the exosome trafficking tag.

[0192] SEQ ID NO: 12: HIV-GAG protein

[0193] MGARASVLSGGELDRWEKIRLRPGGKKKYKRKHIEAASRELERFAVN PGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALDKIEE EQNKSKKKAQQAAADTGHSNQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHP VHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVR MYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCK TILKALGPGATLEEMMTACQGVGGPGHKARVLAEAMSQVTNPATIMIQKGNFRNQR KTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHK GRPGNFLQSRPEPTAPPEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQ

[0194] In certain embodiments, the rhCDKL5 proteins produced by the methods disclosed herein comprise an HIV GAG protein linked to an affinity purification tag selected from: a polyhistidine tag, a polyarginine tag, a glutathione-S-transferase tag, a DYKDDDDK (SEQ ID NO: 29) peptide tag, a streptavidin-binding peptide tag, a peptide tag comprising an amino acid sequence according to any one of SEQ ID NOs: 36 or 37, a calmodulin binding peptide tag, a maltose-binding tag, and a cellulose-binding tag. In certain embodiments, the affinity purification tag is linked by a TEV cleavage site linker at the N-terminus of the CDKL5 sequence. In certain embodiments, the polyhistidine tag is a 6xHIS tag (SEQ ID NO: 21). In certain embodiments, the rhCDKL5 protein comprises an HIV GAG protein or a fragment thereof and a 6xHis tag linked by a linker peptide, and wherein the 6xHis tag is linked to the N-terminus of the rhCDKL5 protein by a TEV protease cleavage site linker. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence set forth in SEQ ID NO: 13. rhCDKL5 for bridging ADA assay

[0195] Development of therapeutics and diagnostics for CDD require raising of CDKL5 specific antibodies that can be used for determining level of CDKL5 in samples from patients suffering from CDD or post-treatment with therapeutics for expressing CDKL5 in CDD patients. However, for detecting the level of such CDKL5 specific antibodies raised in animals requires anti-drug antibody assays (ADA) that utilize detectably labelled CDKL5 proteins that can be used for capturing and detecting the anti-CDKL5 antibodies in samples. However, availability of full-length CDKL5 proteins and their labelling for bridging ADA assays is challenging.

[0196] CDKL5 is a highly disordered protein prone to aggregation and falling out of solution. If subjected to conjugation reagents for random labeling, the CDKL5 protein due to its extensive intrinsic disordered sequences is likely to misfolds and loose the antibody specific epitopes. Alternative methods of labeling that will keep the CDKL5 protein stable are therefore preferred. The methods of producing detectable CDKL5 proteins for the bridging ADA assay approach disclosed herein could make the assay more sensitive, removing the possibility of epitopes being blinded by the addition of a conjugated label.

[0197] The rhCDKL5 proteins produced by the methods of the present disclosure can be used as anti-CDKL5 antibody capturing and detecting partner proteins for bridging ADA assays which can detect anti-CDKL5 antibody in samples. The rhCDKL5 protein for use in the method of detecting anti-CDKL5 antibody disclosed herein can be a CDKL5 protein that comprises a biotinylable peptide tag, GLNDIFEAQKIEWHE (SEQ ID NO: 14) linked to the N- or C-terminus of any of the rhCDKL5 proteins disclosed herein to generate a CDKL5 partner 1 protein. In certain embodiments, the biotinylable peptide tag is located between an intracellular expression tag and the C-terminus of the rhCDKL5 protein. In certain embodiments, the rhCDKL5 protein for use in the method of detecting anti-CDKL5 antibody disclosed herein can be a CDKL5 partner 1 protein, wherein the rhCDKL5 protein comprises an MBP-TEV tag at the C-terminus of the rhCDKL5 protein and a GFP-TEV tag at the N- terminus of the rhCDKL5 protein. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence according to the SEQ ID NO: 18. The rhCDKL5 proteins produced by the methods of the present disclosure, can be biotinylated by linking the biotinylable peptide tag (e.g., biotinylable peptide tag of SEQ ID NO: 14) of the rhCDKL5 protein to a biotin compound. The linking of the biotinylable peptide tag (e.g., biotinylable peptide tag of SEQ ID NO: 14) of the rhCDKL5 protein to a biotin compound can be done in cells of host cell culture that express a biotin ligase enzyme selected from any one of the biotin ligases BirA, BiolD, TurboID, BiolD2, Air ID, MicroID2 and Apex. The biotin ligase enzyme can be E. coli biotin ligase, BirA.

[0198] The rhCDKL5 protein for use in the method of detecting anti-CDKL5 antibody disclosed herein can be a CDKL5 that comprises a peptide tag (e.g., a peptide tag comprising an amino acid sequence of any one of SEQ ID NOs: 30-32 or a fragment thereof (partner 1 peptide tag)) linked to the N- or the C-terminus of any of the rhCDKL5 proteins disclosed herein to generate a CDKL5 partner 1 protein. In certain embodiments, the CDKL5 partner 1protein can bind (e.g., irreversibly or reversibly) to a partner 2 protein comprising an amino acid sequence according to any one of SEQ ID NOs: 33-35 or a fragment thereof. In certain embodiments, the partner 2 protein is labeled with a detectable moiety. The partner 1 protein and the partner 2 protein can be labeling and conjugating partner 1 peptide / partner 2 protein systems known in the art, including but not limited to BioRad SpyCatcher and SpyTag products, Kerafast Biotech SpyTag / SpyCatcher Protein Coupling Reagents or Addgene SpyTag-β-Lactamase-SpyCatcher proteins. In certain embodiments, the partner 1 peptide tag or a fragment thereof can be linked to the N-terminus of a rhCDKL5 protein comprising an amino acid sequence according to SEQ ID NO: 5. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the rhCDKL5 protein linked to a partner 1 peptide tag at the N-terminus, can comprise an amino acid sequence according to SEQ ID NO: 22. In certain embodiments, the partner 1 peptide tag or a fragment thereof can be located between the GFP-TEV linker and the N- terminus of the CDKL5 sequence in the rhCDKL5 protein comprising an amino acid sequence according to SEQ ID NO: 5. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 23.

[0199] In certain embodiments, the partner 1 peptide tag or a fragment thereof can be linked to the C-terminus of a rhCDKL5 protein comprising an amino acid sequence according to SEQ ID NO: 5. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the rhCDKL5 protein linked to a partner 1 peptide tag at the C-terminus, can comprise an amino acid sequence according to SEQ ID NO: 24. In certain embodiments, the partner 1 peptide tag or a fragment thereof can be located between the C-terminus of the CDKL5 sequence and the MBP-TEV linker in the rhCDKL5 protein comprising an amino acid sequence according to SEQ ID NO: 5. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence that is at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 25. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO: 25. Detection, and Affinity Tags

[0200] Various protein tags can be added to recombinant polypeptide sequences to provide additional functional traits. For instance, many tags are known which can be used to detect a particular recombinant polypeptide, i.e., in a sample. Other tags are known which can be used to isolate a particular polypeptide from a sample (i.e., “affinity” or “purification” tags). Still other tags are known which can be used to improve solubility of a particular recombinant polypeptide.

[0201] To improve solubility, detectability, and or purification capability, rhCDKL5 proteins may further comprise cleavable, detection, affinity, and / or solubility tags. Examples of tags suitable for use in rhCDKL5s include, but are not limited to: green fluorescent protein “GFP” tag; maltose binding protein “MBP” tag; polyhistidine tag (such as 6xHIS tag, SEQ ID NO: 21); and / or including variants of any of the foregoing. Persons skilled in the art will appreciate numerous other tags suitable for use in the compositions and methods described herein.

[0202] These tags and other tags can be present at the N-terminus of the rhCDKL5 and / or at the C-terminus of the rhCDKL5. These tags can be present in the rhCDKL5 alone or in combination with other tags or in combination with each other. Persons skilled in the art will appreciate that fusion of one or more of these tags may involve direct fusion of the amino acid sequence of the one or more tags directly together or directly to a rhCDKL5, or alternatively may include appropriate spacers or linkers (such as additional amino acids or other chemical linkers) between the one or more tags and / or between the tags and the rhCDKL5.

[0203] In some cases, it is desirable to be able to remove one or more tags fused to recombinant proteins. Various protein cleavage or enzyme degradable sites are known in the art. Such cleavage sites can be introduced along with tags as described herein to facilitate removal of the tags post-expression and / or post-purification. These cleavage sites can be denoted as cleavage site linkers. An exemplary cleavage site linker useful in the rhCDKL5s described herein is the tobacco etch virus “TEV” protease cleavage site linker, ENLYFQS(SEQ ID NO: 19) or ENLYFQG (SEQ ID NO: 20). Persons skilled in the art will recognize various other cleavage sites or cleavage site linkers that can be substituted in the tagged rhCDKL5 proteins described herein.

[0204] FIGs.2A-2B show various exemplary configurations of intracellular expression tags (e.g., MBP and GFP) linked to a TEV protease cleavage site linker that can be added to the CDKL5 sequence to generate rhCDKL5 proteins according to embodiments of the methods and compositions described herein.

[0205] FIGs. 2C-2E show exemplary configurations of certain secretion signal tagged versions of rhCDKL5 protein according to embodiments of the methods and compositions described herein. In certain exemplary embodiments, the secretable rhCDKL5 proteins of the present disclosure are linked with light chain, heavy chain or hemagglutinin signal sequence tags at the N-terminus and a 6xHIS tag (SEQ ID NO: 21) linked to the C-terminus of the rhCDKL5 protein.

[0206] FIG. 2F shows an exemplary configuration of the exosome trafficable rhCDKL5 protein according to embodiments of the methods and compositions described herein. In certain exemplary embodiments, the exosome trafficable rhCDKL5 protein comprises a HIV-GAG protein linked by a linker peptide to a 6xHIS tag (SEQ ID NO: 21) that is in turn linked to the N-terminus of the rhCDKL5 protein. Methods of Producing Full-Length, Soluble, Non-Denatured rhCDKL5s

[0207] Provided herein are methods of producing recombinant human CDKL5 (rhCDKL5) proteins. In some embodiments, the methods enable reliable production of soluble and full-length rhCDKL5 proteins intracellularly, secreted into the cell culture supernatant or in exosome compartment of cell culture supernatant, of host cell cultures expressing the rhCDKL5 protein. In some embodiments, the methods enable reliable production of non- denatured, soluble and full-length rhCDKL5 proteins.

[0208] The rhCDKL5 proteins produced according to the methods described herein can be any rhCDKL5 proteins. In certain embodiments, the rhCDKL5 protein produced by the methods of the present disclosure comprises a CDKL5 sequence comprising an N-terminal domain kinase domain, and a C-terminal regulatory domain comprising several intrinsically disordered protein (IDP) sequences which further contains an N-terminal nucleus localization signal sequences, NLS1 and a C-terminal nuclear localization signal, NLS2. The CDKL5sequence of the rhCDKL5 produced by the methods of the present disclosure comprise the N- terminal domain kinase domain, and a C-terminal regulatory domain, can be at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. The CDKL5 sequence of the rhCDKL5 produced by the methods of the present disclosure comprise the N- terminal domain kinase domain, and a C-terminal regulatory domain, according to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. Production of intracellular expression signal tagged rhCDKL5

[0209] In some embodiments, the methods of the present disclosure produce a rhCDKL5 protein tagged to at least one intracellular expression tag that facilitates the soluble and stable expression of the rhCDKL5 in the cytoplasm of a host cell transfected with a nucleic acid encoding the tagged rhCDKL5 protein. In some embodiments, the method of the present disclosure for producing a rhCDKL5 protein tagged to an intracellular expression tag comprises: (a) transfecting a host cell culture with a nucleic acid encoding the rhCDKL5 protein; (b) expressing the rhCDKL5 protein in the host cell culture; (c) generating a whole cell lysate by incubating the host cell culture with a lysis buffer; (d) separating soluble fraction comprising the rhCDKL5 protein and insoluble fraction from the whole cell lysate; and (e) isolating the rhCDKL5 protein from the soluble fraction.

[0210] In some embodiments, the at least one intracellular expression tag is linked to the N-terminus and / or the C-terminus of the rhCDKL5 protein. In some embodiments, the intracellular expression tag is selected from a GFP tag, a MBP tag, and a combination thereof. In some embodiments, the rhCDKL5 protein comprises a GFP tag or an MBP tag linked to the N-terminus of the rhCDKL5 protein. In some embodiments, the rhCDKL5 protein comprises a GFP tag or an MBP tag linked to the C-terminus of the rhCDKL5 protein. In some embodiments, the rhCDKL5 protein produced by the method disclosed herein comprise an MBP tag linked to the N-terminus and a GFP-tag linked to the C-terminus of the rhCDKL5 protein. In some embodiments, the rhCDKL5 protein produced by the method disclosed herein comprise a GFP tag linked to the N-terminus and an MBP tag linked to the C-terminus of the rhCDKL5 protein. In some embodiments, the GFP tag and / or the MBP tag are linked to the rhCDKL5 protein by a TEV protease cleavage site linker. In some embodiments, rhCDKL5 linked to the intracellular expression tags produced by the method disclosed herein comprise an amino acid sequence according to SEQ ID NO: 4. In some embodiments, the rhCDKL5linked to the intracellular expression tags produced by the method disclosed herein comprise an amino acid sequence according to SEQ ID NO: 5.

[0211] Generally, rhCDKL5 proteins expressed in host cell cultures will be bound to, embedded in, or strongly associated with the cytoplasm and nucleus of the host cells, necessitating efficient extraction or release from cellular compartments. For the rhCDKL5 proteins to be useful in downstream assays or analysis, the extraction should result in non- denatured rhCDKL5 protein.

[0212] The methods provided herein for extracting these rhCDKL5 proteins generally entail lysing of rhCDKL5-protein-expressing cells, cell fractionation by centrifugation, then release of the soluble fraction containing the rhCDKL5 protein from the insoluble cellular fraction. The methods may further entail subsequent purification of rhCDKL5 protein from the soluble fraction. The methods may further comprise steps to remove any intracellular expression tag, detection, and / or affinity tags present on the rhCDKL5 protein. The disclosed process advantageously produces full-length, soluble and non-denatured rhCDKL5 protein.

[0213] The methods provided herein comprise maintaining the host cell culture under conditions and for an amount of time that is optimal for expression of the rhCDKL5 protein. In some embodiments, the conditions can be maintaining the host cell culture at a temperature of between about 32oC to about 37oC (about 32oC to about 33oC, about 33oC to about 34oC, about 34oC to about 35oC, about 35oC to about 36oC, about 36oC to about 37oC, about 32oC to about 34oC, about 33oC to about 36oC, about 34oC to about 37oC, and all integers including and between 32oC and 37oC). In some embodiments, the host cell culture is maintained at a temperature about 37oC.

[0214] In some embodiments, the methods comprise incubating the rhCDKL5 protein expressing cells with a lysis buffer, preparing a whole cell lysate from the cells, then extracting the soluble fraction containing the rhCDKL5 protein from the insoluble cell membrane fraction by centrifugation.

[0215] Host cells expressing the rhCDKL5 proteins may be prepared by transient transfection of a plasmid DNA encoding the rhCDKL5 protein in a cell suitable for expression of the same. Alternatively, the host cells expressing the rhCDKL5 protein may stably express a transgene encoding the rhCDKL5 protein(s). Methods of producing host cells transientlytransfected with a plasmid DNA and / or stably expressing a transgene of interest will be known by persons skilled in the art.

[0216] Host cells suitable for expressing the rhCDKL5 proteins comprise HEK293 cells (such as Expi293FTMcells from ThermoFisher Scientific), HEK293T cells, BHK cells, CHO cells, COS cells, and / or HeLa cells. Once transfected or modified to stably express the rhCDKL5, the host cell culture can be maintained for a duration of time and under conditions appropriate for protein expression. Culture conditions which can be optimized according to routine methodology include selection and supplementation of growth media, culture format (suspension versus adherent culture, for example), incubation time, incubation temperature, pH, dissolved oxygen levels, and the like.

[0217] The host cell culture can be maintained under conditions for expressing the rhCDKL5 protein, e.g., for at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 30 hours, at least about 36 hours, at least about 42 hours, at least about 48 hours, at least about 54 hours, at least about 60 hours, at least about 66 hours, at least about 72 hours, at least about 78 hours, at least about 84 hours, at least about 90 hours, or at least about 96 hours. In one embodiment, the host cell culture can be maintained under conditions for expressing the rhCDKL5 protein, e.g., for about 48 hours to about 72 hours.

[0218] The percentage of live cells in a host cell culture is expected to decrease over time, which may reduce the ability to recover expressed protein over time. In some embodiments, the host cell culture can be maintained for an amount of time until the percentage of live cells of the host cell culture post-transfection with the nucleic acid encoding the rhCDKL5 protein is at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, or at least about 50%. The percentage of live cells present in a cell culture can be expressed as viability of the cell culture. Viability may be evaluated, e.g., by microscopy-based assays, colorimetry-based assays, or flow-cytometry-based assays, or combinations thereof.

[0219] In transiently transfected host cell cultures, the percentage of transfected cells may decrease over time, which may reduce the ability to recover expressed protein over time. In some embodiments, the host cell culture can be maintained for an amount of time until the percentage of transfected cells in the host cell culture post-transfection with the nucleic acidencoding the rhCDKL5 protein is at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, or at least about 50%. The percentage of transfected cells may be evaluated by microscopy-based assays, colorimetry-based assays, or flow- cytometry-based assays, or combinations thereof.

[0220] The expression of the rhCDKL5 protein in the transfected host cells can be increased by maintaining the host cell culture in presence of at least one expression enhancer selected from aromatic carboxylic acids, aliphatic carboxylic acids, hydroxamic acids, acetamides, sodium butyrate or valproic acid. The aromatic carboxylic acids can be any one of phthalic acid, isophthalic acid, and terephthalic acid. The hydroxamic acid can be any one of vorinostat, belinostat, panobinostat, valproic acid, sodium butyrate, and trichostatin A. The expression enhancer can be sodium butyrate or valproic acid. In certain embodiments, the at least one expression enhancer comprises an aromatic carboxylic acid, an aliphatic carboxylic acid, a hydroxamic valproic acid, a hydroxamic derivative of valproic acid, an acetamide, sodium butyrate, or a derivative of any of the foregoing.

[0221] Following culturing of the host cells, the cells can be contacted with a lysis buffer to lyse the cells in the host cell culture. The lysis buffer may comprise a detergent. The detergent may be a non-ionic detergent, a cationic detergent, an anionic detergent or a zwitterionic detergent. Numerous detergents suitable for lysing cultured host cells are known. In some embodiments, the lysis buffer can be one or more of Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), octylphenoxypolyethoxyethanol (Nonidet P-40), and ammonium-chloride-potassium (ACK) lysis buffer.

[0222] The lysis buffer can further comprise one or more protease inhibitor(s) selected from phenylmethylsulfonyl fluoride (PMSF), 4-(2-Aminoethyl)-benzenesulfonylfluoride hydrochloride (AEBSF), Aprotinin, Benzamidine, Bafilomycin A, ethylenediamine-tetraacetic acid disodium salt (EDTA), aprotinin, epoxide, bestatin, MG-132, Pepstatin A, Lactacystin, Leupeptin and alpha-2 macroglobulin or a combination thereof. The lysis buffer can further comprise one or more salts selected from sodium chloride, sodium orthovanadate, sodium fluoride, magnesium chloride and sodium acetate. The protease inhibitor can be any commercial protease inhibitor cocktails known in the art. The lysis buffer can further comprise a chelating agent. Chelating agents are well known in the art. In some embodiments, the lysis buffer comprises a chelating agent selected from ethylenediamine-tetraacetic acid disodiumsalt (EDTA); ethyleneglycol-O, O’-bis(2-aminoethyl)-N, N, N’, N’-tetraacetic acid (EGTA); N-(2-Hydroxyethyl)ethylenediamine-N, N’, N’-triacetic acid trisodium salt (HEDTA); nitrilotriacetic acid (NTA); and potassium citrate.

[0223] The cells in the host cell culture can be contacted with the lysis buffer for an amount of time sufficient to generate a whole cell lysate. In some embodiments, the cells can be contacted with the lysis buffer for about 10 min to about 60 min (e.g., about 10 min to about 20 min, about 20 min to about 40 min, about 30 min to about 40 min, about 40 min to about 60 min, and all integers including and between 10 min to 60 min). In some embodiments, the cells can be contacted with the lysis buffer for about 10 min, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes.

[0224] The cells can be contacted with the lysis buffer and subjected to sonication or homogenization, for an amount of time sufficient to generate a whole cell lysate. In certain embodiments, the cells can be contacted with the lysis buffer and subjected to homogenization, for an amount of time sufficient to generate a whole cell lysate.

[0225] The whole cell lysate can be centrifuged to generate at least one soluble cell lysate fraction comprising the cytoplasmic fraction of the cells from the insoluble fraction (cell membrane and organelles). Centrifugation can be performed, e.g., at about 1000×g to about 12000×g, for example, at about 1000×g, about 1100×g, about 1200×g, about 1300×g, about 1400×g, about 1500×g, about 2000×g, about 2500×g, about 3000×g, about 3500×g, about 4000×g, about 4500×g, about 5000×g, about 5500×g, about 6000×g, about 6500×g, about 7000×g, about 7500×g, about 8000×g, about 8500×g, about 9000×g, about 9500×g, about 10000×g, about 11000×g, or about 12000×g. The centrifugation can be performed, e.g., for about 5 minutes to about 1 hour, for example about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes.

[0226] The methods of producing rhCDKL5 proteins described herein may further comprise steps to isolate and / or purify the released rhCDKL5 protein. Exemplary methods for isolating and / or purifying exogenously expressed recombinant protein are known. In some embodiments, affinity-based chromatography may be used to isolate and / or purify the rhCDKL5 protein from the soluble fraction. For instance, an affinity-based chromatography resin may be used to capture the intracellular expression tagged rhCDKL5 protein produced bythe methods of the present disclosure, from a solution comprising the released rhCDKL5 protein. The affinity may be immunoaffinity-mediated, such as with an antibody or antigen- binding fragment specific to an antigen present in the rhCDKL5 protein. Alternatively, the chromatography-based resin may have an affinity to a particular affinity / detection / intracellular expression tag present on the rhCDKL5 protein. Examples of chromatography-based resins that can be used to purify rhCDKL5 proteins produced by the method of the present disclosure include, anti-GFP antibody chromatography resin (as in case of GFP-tagged rhCDKL5 protein), reduced-glutathione chromatography resin (as in the case of GST-tagged rhCDKL5 protein), Ni-NTA chromatography resin (as in the case of a HIS-tagged rhCDKL5 protein), dextrin crosslinked to agarose affinity resin (dextrin-sepharose chromatography resin) (as in the case of MBP-tagged rhCDKL5 protein), or an amylose chromatography resin (as in the case of MBP-tagged rhCDKL5 protein). In one embodiment, the affinity-based chromatography resin to capture the intracellular expression tagged rhCDKL5 protein produced by the methods of the present disclosure from a solution is a dextrin crosslinked to agarose affinity resin.

[0227] Certain embodiments of the methods for producing rhCDKL5 proteins described herein comprise culturing Expi293F cells transiently transfected with a plasmid DNA encoding the rhCDKL5 protein and harvesting the cells, e.g., by centrifugation to generate a cell pellet. Post-harvest of the Expi293 cells, the cell pellet may be lysed in a lysis buffer. The cell pellet may additionally or alternatively be sonicated or homogenized to disrupt cell membranes. Next, lysed cells may be centrifuged to obtain a post-lysis cell pellet and supernatant. As an example, centrifugation may be used to generate a post-solubilization supernatant and an insoluble pellet. The supernatant post-solubilization may be further filtered and loaded onto a chromatography column, e.g., a dextrin crosslinked to agarose affinity resin column, using step elution at pH 8-8.5. The loaded chromatography column may be washed and eluted to obtain elution fractions which can be analyzed for presence of the purified rhCDKL5 protein. Western blotting using an antibody specific to the GFP tag, MBP tag or CDKL5 protein or other suitable techniques may be used to detect the rhCDKL5 protein at any step of the process. Production of secretion signal tagged rhCDKL5

[0228] In some embodiments, the methods of the present disclosure are for producing a rhCDKL5 protein tagged to one or more secretion signal tags that facilitate the soluble andstable expression and secretion of the rhCDKL5 in the cell culture supernatant / milieu of a host cell transfected with a nucleic acid encoding the secretion signal tagged rhCDKL5 protein. In some embodiments, the methods of the present disclosure for producing a rhCDKL5 protein tagged to at least one secretion signal tags comprises: (a) transfecting a host cell culture with a nucleic acid encoding the rhCDKL5 protein; (b) expressing the rhCDKL5 protein in the host cell culture; and (c) separating a supernatant from the host cell culture, wherein the supernatant comprises the expressed rhCDKL5 protein.

[0229] In some embodiments, the at least one secretion signal tag is linked to the N- terminus and / or the C-terminus of the rhCDKL5 protein. In certain embodiments, the at least one secretion signal tag is selected from (a) a kappa light chain signal sequence tag; MDMRVPAQLLGLLLLWLPGAKC (SEQ ID NO: 6); (b) a heavy chain signal sequence tag; MGVKVLFALICIAVAEA (SEQ ID NO: 7); and (c) a hemagglutinin signal sequence tag MKTIIALSYIFCLVFA (SEQ ID NO: 8). In certain embodiments, the secretion signal tag linked rhCDKL5 protein produced by the method of the present disclosure can comprise an affinity purification tag linked to the N-terminus of the rhCDKL5 protein when the rhCDKL5 protein has the at least one secretion signal tags linked to the C-terminus of the rhCDKL5 protein. In certain embodiments, the secretion signal tag linked rhCDKL5 protein produced by the method of the present disclosure can comprise an affinity purification tag linked to the C- terminus of the rhCDKL5 protein when the rhCDKL5 protein has the at least one secretion signal tag linked to the N-terminus of the rhCDKL5 protein. The affinity purification tag can be any one of a polyhistidine tag, a polyarginine tag, a glutathione-S-transferase tag, a FLAG®tag (DYKDDDDK (SEQ ID NO: 29), a streptavidin-binding peptide tag, a peptide tag comprising an amino acid sequence according to any one of SEQ ID NOs: 36 or 37, a calmodulin binding peptide tag, a maltose-binding tag and a cellulose-binding tag. In some embodiments, the affinity purification tag is a polyhistidine tag, for example, a 6xHIS tag (SEQ ID NO: 21). In some embodiments, the method of the present disclosure for producing a rhCDKL5 protein tagged to at least one secretion signal tag, further comprises isolating / purifying the rhCDKL5 protein from the supernatant using any one of a nickel- nitrilotriacetic acid (Ni-NTA) chromatography resin, a cross linked dextran chromatography resin, an immobilized glutathione resin, an anti-DYKDDDDK (SEQ ID NO: 29) peptide tag antibody resin, a streptavidin resin, a biotin resin, an immobilized chitin resin, a cellulose resin, a maltose resin, and a calmodulin resin. In certain embodiment, the method of the presentdisclosure for producing a rhCDKL5 protein tagged to at least one secretion signal tag, further comprises isolating / purifying the rhCDKL5 protein from the supernatant using a nickel- nitrilotriacetic acid (Ni-NTA) chromatography resin.

[0230] In some embodiments, the secretion signal tag linked rhCDKL5 protein produced by the method of the present disclosure comprises a kappa light chain signal sequence tag linked to the N-terminus of the rhCDKL5 protein and a 6xHIS tag (SEQ ID NO: 21) linked to the C-terminus of the rhCDKL5 protein. In certain embodiments, the secretion signal tag linked rhCDKL5 protein produced by the method of the present disclosure comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the secretion signal tag linked rhCDKL5 protein produced by the method of the present disclosure comprises an amino acid sequence according to SEQ ID NO: 9. In some embodiments, the secretion signal tag linked rhCDKL5 protein produced by the method of the present disclosure comprises a heavy chain signal sequence tag linked to the N-terminus of the rhCDKL5 protein and a 6xHIS tag (SEQ ID NO: 21) linked to the C-terminus of the rhCDKL5 protein. In certain embodiments, the secretion signal tag linked rhCDKL5 protein produced by the method of the present disclosure comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the secretion signal tag linked rhCDKL5 protein produced by the method of the present disclosure comprises an amino acid sequence according to SEQ ID NO: 10. In some embodiments, the secretion signal tag linked rhCDKL5 protein produced by the method of the present disclosure comprises a hemagglutinin signal sequence tag linked to the N-terminus of the rhCDKL5 protein and a 6xHIS tag (SEQ ID NO: 21) linked to the C-terminus of the rhCDKL5 protein. In certain embodiments, the secretion signal tag linked rhCDKL5 protein produced by the method of the present disclosure comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the secretion signal tag linked rhCDKL5 protein produced by the method of the present disclosure comprises an amino acid sequence according to SEQ ID NO: 11.

[0231] Generally, rhCDKL5 proteins linked to at least one secretion signal tag will be expressed in host cell cultures and secreted into the host cell culture milieu / supernatant, necessitating efficient separation of the cell culture supernatant containing the rhCDKL5 protein from the cells, and subsequent purification of the rhCDKL5 protein from the cell culture supernatant. For the rhCDKL5 proteins to be useful in downstream assays or analysis, the purification should result in non-denatured rhCDKL5 protein. In certain embodiments, separating the supernatant comprising the expressed rhCDKL5 protein from the host cell culture is repeated or is conducted continuously throughout the duration of expressing the rhCDKL5 protein in the host cell culture

[0232] The methods provided herein for producing the rhCDKL5 proteins linked to at least one secretion signal tag comprises maintaining the host cell culture expressing the rhCDKL5 protein at about 32oC to about 37oC (about 32oC to about 33oC, about 33oC to about 34oC, about 34oC to about 35oC, about 35oC to about 36oC, about 36oC to about 37oC, about 32oC to about 34oC, about 33oC to about 36oC, about 34oC to about 37oC, and all integers including and between 32oC and 37oC). In certain embodiments, the host cell culture expressing the rhCDKL5 protein is maintained at about 37oC. The maintaining of the host cell culture expressing the rhCDKL5 protein is done in any cell culture media known in the art for culturing AAV expressing host cells including but not limited to Eagle's Minimum Essential Medium (EMEM) or Dulbecco's Modified Eagle's Medium (DMEM).

[0233] The methods provided herein for producing the rhCDKL5 proteins linked to at least one secretion signal tag generally entails cell culture supernatant isolation by centrifugation of the rhCDKL5 expressing cell culture, containing the soluble rhCDKL5 protein from the insoluble cellular fraction. The methods may further entail subsequent purification of rhCDKL5 protein from the supernatant fraction. The methods may further comprise steps to remove any secretion signal tags, detection, and / or affinity tags present on the rhCDKL5 protein. The disclosed process advantageously produces full-length, soluble and non-denatured rhCDKL5 protein. The methods provided herein for producing at least one secretion signal tagged rhCDKL5 proteins comprise detecting presence of rhCDKL5 protein in the host cell culture supernatant and separating the supernatant from the host cell culture if rhCDKL5 protein is detected in the supernatant. In some embodiments, rhCDKL5 protein in the host cell culture supernatant is detected using Western blotting (by probing with anti- CDKL5 protein or signal sequence tag specific antibody, e.g., anti-HA antibody or anti-kappachain antibody) or capillary electrophoresis with laser-induced fluorescence detection (if rhCDKL5 protein is linked to a fluorescent peptide tag).

[0234] Host cells (host cell culture) expressing the rhCDKL5 proteins may be prepared by transient transfection of a plasmid DNA encoding the rhCDKL5 protein linked to one or more secretion signal tag, in a cell suitable for expression of the same. Alternatively, the host cells (host cell culture) expressing the rhCDKL5 protein linked to one or more secretion signal tag, may stably express a transgene encoding the rhCDKL5 protein(s). Methods of producing host cells transiently transfected with a plasmid DNA and / or stably expressing a transgene of interest will be known by persons skilled in the art.

[0235] Host cells suitable for expressing the rhCDKL5 protein linked to at least one secretion signal tag include HEK293 cells (such as Expi293FTM cells from ThermoFisher Scientific), HEK293T cells, BHK cells, CHO cells, COS cells, and / or HeLa cells. Once transfected or modified to stably express the rhCDKL5 protein linked to at least one secretion signal tag, the host cell culture can be maintained for a duration of time and under conditions appropriate for protein expression. Culture conditions which can be optimized according to routine methodology include selection and supplementation of growth media, culture format (suspension versus adherent culture, for example), incubation time, incubation temperature, pH, dissolved oxygen levels, and the like.

[0236] The host cell culture can be maintained under conditions adequate to express the rhCDKL5 protein, for a duration up to 7 days (e.g., up to 1 day, up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days, up to 7 days).

[0237] The methods provided herein comprise maintaining the host cell culture under conditions and for an amount of time that is optimal for expression of the rhCDKL5 protein. In some embodiments, the conditions can be maintaining the host cell culture at a temperature of between about 32oC to about 37oC (about 32oC to about 33oC, about 33oC to about 34oC, about 34oC to about 35oC, about 35oC to about 36oC, about 36oC to about 37oC, about 32oC to about 34oC, about 33oC to about 36oC, about 34oC to about 37oC, and all integers including and between 32oC and 37oC). In some embodiments, the host cell culture is maintained at a temperature about 37oC.

[0238] The percentage of live cells in a host cell culture is expected to decrease over time, which may reduce the ability to recover the rhCDKL5 linked to one or more secretionsignal tags of the present disclosure, from host cell culture supernatant over time. In some embodiments, the host cell culture can be maintained for an amount of time until the percentage of live cells of the host cell culture post-transfection with the nucleic acid encoding the rhCDKL5 protein is at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, or at least about 50%. The percentage of live cells present in a cell culture can be expressed as viability of the cell culture. Viability may be evaluated, e.g., by microscopy-based assays, colorimetry-based assays, or flow-cytometry-based assays, or combinations thereof.

[0239] In transiently transfected host cell cultures, the percentage of transfected cells may decrease over time, which may reduce the ability to recover the rhCDKL5 linked to one or more secretion signal tags of the present disclosure, from host cell culture supernatant over time. In some embodiments, the host cell culture can be maintained for an amount of time until the percentage of transfected cells in the host cell culture post-transfection with the nucleic acid encoding the rhCDKL5 protein is at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, or at least about 50%. The percentage of transfected cells may be evaluated by microscopy-based assays, colorimetry-based assays, or flow- cytometry-based assays, or combinations thereof.

[0240] The host cell culture expressing the rhCDKL5 linked to at least one secretion signal tag of the present disclosure, can be centrifuged to generate at least one soluble supernatant fraction comprising the rhCDKL5 protein from the insoluble cellular fraction. Centrifugation can be performed, e.g., at about 1000×g to about 12000×g, for example, at about 1000×g, about 1100×g, about 1200×g, about 1300×g, about 1400×g, about 1500×g, about 2000×g, about 2500×g, about 3000×g, about 3500×g, about 4000×g, about 4500×g, about 5000×g, about 5500×g, about 6000×g, about 6500×g, about 7000×g, about 7500×g, about 8000×g, about 8500×g, about 9000×g, about 9500×g, about 10000×g, about 11000×g, or about 12000×g. The centrifugation can be performed, e.g., for about 5 minutes to about 1 hour, for example about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes.

[0241] The methods of producing rhCDKL5 protein linked to at least one secretion signal tag described herein, may further comprise steps to separate / purify the released rhCDKL5 protein from the supernatant isolated from the host cell culture expressing therhCDKL5 protein. In some embodiments, the method comprises separating the supernatant comprising the expressed rhCDKL5 from the host cell culture. In some embodiments, the step of separating the supernatant comprising the expressed rhCDKL5 from the host cell culture is repeated or is conducted continuously throughout the duration of expressing the rhCDKL5 protein in the host cell culture. Exemplary methods for separating / purifying exogenously expressed recombinant protein are known in the art. In some embodiments, affinity chromatography may be used to purify the rhCDKL5 protein linked to one or more secretion signal tags described herein. For instance, an affinity chromatography resin may be used to capture the intracellular expression tagged rhCDKL5 protein produced by the method of the present disclosure, from a host cell culture supernatant comprising the released rhCDKL5 protein. The affinity may be immunoaffinity-mediated, such as with an antibody or antigen- binding fragment specific to an antigen present in the rhCDKL5 protein. Alternatively, the chromatography resin may have an affinity to a particular affinity / detection / secretion signal tag present on the rhCDKL5 protein. Examples of chromatography resins that can be used to purify rhCDKL5 proteins produced by the method of the present disclosure including, a Ni- NTA chromatography resin (as in the case of a polyhistidine tag linked rhCDKL5 protein); a cross linked dextran chromatography resin (as in the case of a polyarginine tag linked rhCDKL5 protein); an immobilized glutathione resin (as in the case of a glutathione-S- transferase tag linked rhCDKL5 protein); an anti-FLAG antibody resin (as in the case of a FLAG tag linked rhCDKL5 protein); a biotin resin (as in the case of a streptavidin-binding peptide tag linked rhCDKL5 protein); a cellulose resin (as in the case of a calmodulin binding peptide tag linked rhCDKL5 protein); a maltose resin (as in the case of a maltose-binding tag linked rhCDKL5 protein); and a calmodulin resin (as in the case of a cellulose-binding tag linked rhCDKL5 protein). In some embodiments, the cross linked dextran chromatography resin is a SP Sephadex™ resin.

[0242] Certain embodiments of the methods for producing rhCDKL5 proteins linked to at least one secretion signal tag described herein, comprise culturing Expi293F cells transiently transfected with a plasmid DNA encoding the rhCDKL5 protein linked to at least one secretion signal tag and harvesting the cells, e.g., by centrifugation to generate supernatant separated from a cell pellet. Post-harvest of the Expi293 cells, the supernatant may be filtered or subjected to buffer exchange to remove impurities including salts. The supernatant with or without buffer exchange may be further filtered and loaded onto a chromatography column,e.g., Ni-NTA column chromatography resin, using step elution at pH 7.5-8.0. The loaded chromatography column may be washed and eluted to obtain elution fractions which can be analyzed for presence of the purified rhCDKL5 protein linked to at least one secretion signal tag. Western blotting using an antibody specific to the CDKL5 protein, HIS tag or any other detectable tag or other suitable techniques may be used to detect the rhCDKL5 protein at any step of the process. Production of an exosome trafficking tag linked rhCDKL5

[0243] In some embodiments, the methods of the present disclosure are for producing a rhCDKL5 protein tagged to an exosome trafficking tag that facilitates transport of the rhCDKL5 protein into exosomes released in the cell culture supernatant / milieu of a host cell transfected with a nucleic acid encoding the secretion signal tagged rhCDKL5 protein. In some embodiments, the method of the present disclosure for producing a rhCDKL5 protein tagged to an exosome trafficking tag comprises: transfecting a host cell culture with a nucleic acid encoding the rhCDKL5 protein; expressing the rhCDKL5 protein in the host cell culture; and separating a supernatant from the host cell culture, wherein the supernatant comprises the exosomes comprising the expressed rhCDKL5 protein.

[0244] In some embodiments, the exosome trafficking tag is an exosome-targeting viral peptide or a fragment thereof. In some embodiments, the exosome trafficking tag is selected from an HIV GAG protein or a fragment thereof, an HIV NEF peptide, an HTLV-1 transactivator protein (Tax), an HCV core protein, and an HCV glycoprotein E2. In some embodiments, the exosome trafficking tag is an HIV GAG protein or a fragment thereof.

[0245] In some embodiments, the exosome trafficking tag is linked to the N-terminus or the C-terminus of the rhCDKL5 protein. In some embodiments, the HIV GAG protein or a fragment thereof is linked to the N-terminus of any of the rhCDKL5 proteins disclosed herein. In certain embodiments, the exosome trafficking tag linked rhCDKL5 protein further comprises an affinity purification tag. In certain embodiments, the affinity purification tag is selected from a polyhistidine tag, a polyarginine tag, a glutathione-S-transferase tag, a DYKDDDDK (SEQ ID NO: 29) peptide tag, a streptavidin-binding peptide tag, a peptide tag comprising an amino acid sequence according to any one of SEQ ID NOs: 36 or 37, a calmodulin binding peptide tag, a maltose-binding tag, and a cellulose-binding tag.

[0246] In certain embodiments, the exosome trafficking tag linked rhCDKL5 protein further comprises an affinity purification tag, wherein the exosome trafficking tag is a HIV GAG protein or a fragment thereof and the affinity purification tag is a 6xHIS tag (SEQ ID NO: 21), wherein the exosome trafficking tag and the affinity purification tag are linked by a linker peptide, and wherein the affinity purification tag is linked to the N-terminus of the rhCDKL5 protein by a TEV protease cleavage site linker. In certain embodiments, the rhCDKL5 protein linked to a HIV GAG protein and a 6xHIS tag (SEQ ID NO: 21) produced by the method of the present disclosure comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 13. The rhCDKL5 protein linked to a HIV GAG protein and a 6xHIS tag (SEQ ID NO: 21) produced by the method of the present disclosure comprises an amino acid sequence according to SEQ ID NO: 13.

[0247] Generally, rhCDKL5 proteins linked to an exosome trafficking tag will be expressed in host cell cultures and secreted into the cell culture milieu / supernatant, necessitating efficient separation of the cell culture supernatant comprising the rhCDKL5 protein comprising exosomes from the cells, followed by isolation of the rhCDKL5 protein comprising exosome from the supernatant, and subsequent lysis of the exosomes and purification of the rhCDKL5 protein. For the rhCDKL5 proteins to be useful in downstream assays or analysis, the purification should result in non-denatured rhCDKL5 protein. In certain embodiments, separating the supernatant comprising the expressed rhCDKL5 protein from the host cell culture is repeated or is conducted continuously throughout the duration of expressing the rhCDKL5 protein in the host cell culture.

[0248] The methods provided herein for producing the rhCDKL5 proteins linked to an exosome trafficking tag comprises maintaining the host cell culture expressing the rhCDKL5 protein at about 32oC to about 37oC. In some embodiments, the host cell culture expressing the rhCDKL5 protein linked to an exosome trafficking condition is maintained at a temperature of between about 32oC to about 37oC (about 32oC to about 33oC, about 33oC to about 34oC, about 34oC to about 35oC, about 35oC to about 36oC, about 36oC to about 37oC, about 32oC to about 34oC, about 33oC to about 36oC, about 34oC to about 37oC, and all integers including and between 32oC and 37oC). In certain embodiments, the host cell culture expressing the rhCDKL5 protein is maintained at about 37oC. The maintaining of the host cell cultureexpressing the rhCDKL5 protein linked to an exosome trafficking tag is done in any cell culture media known in the art for culturing AAV expressing host cells including but not limited to Eagle's Minimum Essential Medium (EMEM) or Dulbecco's Modified Eagle's Medium (DMEM).

[0249] The methods provided herein for producing the rhCDKL5 proteins linked to an exosome trafficking tag generally entail cell culture supernatant isolation by centrifugation of the rhCDKL5 expressing cell culture, comprising exosome loaded with the rhCDKL5 protein from the cellular fraction. The methods may further entail subsequent purification of rhCDKL5 protein from the exosome comprising supernatant fraction by first isolating the exosomes from the supernatant fraction by combination of ultracentrifugation and magnetic bead-based exosome specific affinity purification. The magnetic bead-based exosome specific affinity purification can include reagents known in the art, including but not limited to magnetic beads- based CD9, CD63, CD81, or EpCam-specific reagent, or combine the Streptavidin reagent with a biotinylated antibody binding to a exosome specific surface antigen. The methods may further comprise steps to remove any exosome trafficking tags, detection, and / or affinity tags present on the rhCDKL5 protein. The disclosed process advantageously produces full-length, soluble and non-denatured rhCDKL5 protein.

[0250] The methods provided herein for producing rhCDKL5 proteins linked to an exosome trafficking tag comprise detecting presence of rhCDKL5 protein in a sample of the host cell culture supernatant and separating the supernatant from the host cell culture, followed by isolation of exosomes, if rhCDKL5 protein is detected in the supernatant sample. In some embodiments, rhCDKL5 protein in the host cell culture supernatant sample is detected using Western blotting (by probing with anti-CDKL5 protein or exosome trafficking tag specific antibody, e.g., anti HIB GAG peptide chain antibody).

[0251] Host cells expressing the rhCDKL5 proteins may be prepared by transient transfection of a plasmid DNA encoding the rhCDKL5 protein linked to the exosome trafficking tag, in a cell suitable for expression of the same. Alternatively, the host cells expressing the rhCDKL5 protein linked to an exosome trafficking tag, may stably express a transgene encoding the rhCDKL5 protein(s). Methods of producing host cells transiently transfected with a plasmid DNA and / or stably expressing a transgene of interest will be known by persons skilled in the art.

[0252] Host cells suitable for expressing the rhCDKL5 protein linked to an exosome trafficking tag include HEK293 cells (such as Expi293FTM cells from ThermoFisher Scientific), HEK293T cells, BHK cells, CHO cells, COS cells, and / or HeLa cells. Once transfected or modified to stably express the rhCDKL5 protein linked to the exosome trafficking tag, the host cell culture can be maintained for a duration of time and under conditions appropriate for protein expression. Culture conditions which can be optimized according to routine methodology include selection and supplementation of growth media, culture format (suspension versus adherent culture, for example), incubation time, incubation temperature, pH, dissolved oxygen levels, and the like.

[0253] The host cell culture can be maintained under conditions adequate to express the rhCDKL5 protein linked to the exosome trafficking tag, for a duration up to 7 days (e.g., up to 1 day, up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days, up to 7 days).

[0254] The percentage of live cells in a host cell culture is expected to decrease over time, which may reduce the ability to recover the rhCDKL5 linked to an exosome trafficking tag of the present disclosure, from host cell culture supernatant over time. In some embodiments, the host cell culture can be maintained for an amount of time until the percentage of live cells of the host cell culture post-transfection with the nucleic acid encoding the rhCDKL5 protein linked to the exosome trafficking tag is at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, or at least about 50%. The percentage of live cells present in a cell culture can be expressed as viability of the cell culture. Viability may be evaluated, e.g., by microscopy-based assays, colorimetry-based assays, or flow-cytometry-based assays, or combinations thereof.

[0255] In transiently transfected host cell cultures, the percentage of transfected cells may decrease over time, which may reduce the ability to recover the rhCDKL5 linked to an exosome trafficking tag of the present disclosure, from exosomes in the host cell culture supernatant over time. In some embodiments, the host cell culture can be maintained for an amount of time until the percentage of transfected cells in the host cell culture post-transfection with the nucleic acid encoding the rhCDKL5 protein linked to the exosome trafficking tag is at least about 95%, at least about 90%, at least about 80%, at least about 70%, at least about 60%, or at least about 50%. The percentage of transfected cells may be evaluated by microscopy-based assays, colorimetry-based assays, or flow-cytometry-based assays, or combinations thereof.

[0256] The host cell culture expressing the rhCDKL5 linked to an exosome trafficking tag of the present disclosure, can be centrifuged to generate a supernatant fraction comprising the exosomes containing the rhCDKL5 protein from the insoluble cellular fraction. Centrifugation to separate the exosome containing supernatant fraction from the insoluble cellular fraction can be performed, at about 300×g to about 1000×g, for example, at about 300×g, about 400×g, about 500×g, about 600×g, about 700×g, about 800×g, about 900×g, or about 1000×g. The centrifugation can be performed, e.g., for about 5 minutes to about 1 hour, for example about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes. The centrifugation can be performed at about 4oC.

[0257] The supernatant containing the exosomes can be further centrifuges to obtain a pellet fraction containing the exosomes by ultracentrifugation at about 50,000×g to about 100,000×g, for example, about 50,000×g, about 60,000×g, about 70,000×g, about 80,000×g, about 90,000×g or about 100,000×g. The ultracentrifugation can be performed for about 1 hour to 2 hours, for example, about 1 hour, about 1.5 hours or about 2 hours. The ultracentrifugation can be performed at about 4oC.

[0258] The pellet containing the exosomes can be filtered and concentrated in a suspension using any filtration device known in the art, including but not limited to ultracellulose, biomax or centricon. The exosomes can be purified from the filtered and concentrated suspension using any technique known in the art, including but not limited to OptiPrep™ density gradient purification, ExoQuick™ precipitation or Exo-spin™ precipitation. The methods may further entail subsequent purification of rhCDKL5 protein from the exosome containing supernatant fraction by first isolating the exosomes from the supernatant fraction by combination of ultracentrifugation and magnetic bead-based exosome specific affinity purification. The magnetic bead-based exosome specific affinity purification can include reagents known in the art, including but not limited to magnetic beads-based CD9, CD63, CD81, or EpCam-specific reagent, or combine the Streptavidin reagent with a biotinylated antibody binding to a exosome specific surface antigen. The methods may further comprise steps to remove any exosome trafficking tags, detection, and / or affinity tags present on the rhCDKL5 protein. The disclosed process advantageously produces full-length, soluble and non-denatured rhCDKL5 protein.

[0259] The method of producing rhCDKL5 protein linked to an exosome trafficking tag described herein, further comprises contacting the purified exosomes with a lysis buffer for an amount of time sufficient to release the rhCDKL5 protein. The lysis buffer can be any exosome lysis buffer known in the art, including but not limited to radioimmunoprecipitation assay (RIPA) buffer, Tris-SDS based buffer, Tris-Triton based buffer, GuHCl based buffer or urea-thiourea based buffer. The contacting of the purified exosomes with a lysis buffer, may be accompanied with sonication or homogenization. The exosome can be purified post- contacting with the lysis buffer by centrifuging the lysis buffer-exosome mixture at about 10,000×g to about 20,000×g, for example, about 10,000×g, about 12,000×g, about 14,000×g, about 16,000×g, about 18,000×g or about 20,000×g, for about 1 hour to about 2 hours, for example, about 1 hour, about 1.5 hours or about 2 hours, at about 4oC, to generate a supernatant containing the rhCDKL5 protein and an insoluble membrane pellet fraction.

[0260] The method of producing rhCDKL5 protein linked to an exosome trafficking tag described herein, further comprises purifying the rhCDKL5 protein linked to the exosome trafficking tag. For instance, an affinity chromatography resin may be used to capture the exosome trafficking tag linked rhCDKL5 protein produced by the method of the present disclosure, from the lysate obtained from exosomes isolated form cells expressing the rhCDKL5 protein. The affinity may be immunoaffinity-mediated, such as with an antibody or antigen-binding fragment specific to an antigen present in the rhCDKL5 protein. Alternatively, the chromatography resin may have an affinity to a particular affinity / detection / secretion signal tag present on the rhCDKL5 protein. Examples of chromatography resins that can be used to purify rhCDKL5 proteins produced by the method of the present disclosure including, a Ni- NTA chromatography resin (as in the case of a polyhistidine tag linked rhCDKL5 protein); a SP-Sephadex chromatography resin (as in the case of a polyarginine tag linked rhCDKL5 protein); an immobilized glutathione resin (as in the case of a glutathione-S-transferase tag linked rhCDKL5 protein); an anti-DYKDDDDK (SEQ ID NO: 29) peptide tag antibody resin (as in the case of a FLAG (DYKDDDDK, SEQ ID NO: 29) tag linked rhCDKL5 protein); a biotin resin (as in the case of a streptavidin-binding peptide tag linked rhCDKL5 protein); a cellulose resin (as in the case of a calmodulin binding peptide tag linked rhCDKL5 protein); a maltose resin (as in the case of a maltose-binding tag linked rhCDKL5 protein); and a calmodulin resin (as in the case of a cellulose-binding tag linked rhCDKL5 protein). In some embodiments, the cross linked dextran chromatography resin is a SP Sephadex™ resin.

[0261] Certain embodiments of the methods for producing rhCDKL5 proteins linked to an exosome trafficking tag described herein, comprise culturing Expi293F cells transiently transfected with a plasmid DNA encoding the rhCDKL5 protein linked to the exosome trafficking tag and harvesting the cells, e.g., by centrifugation to generate supernatant containing the exosomes produced from the cells separated from a cell pellet. Post-harvest of the Expi293 cells, the supernatant may be filtered or subjected to filtration to remove impurities including salts and larger vesicles, using the methods described herein. The exosomes may be purified from the supernatant by using any of the exosome purification methods known in the art, including exosome specific affinity-based purification using magnetic beads, as described herein. The exosomes so purified may be lysed using a suitable lysis buffer known in the art and described herein, followed by centrifugation to generate a supernatant containing the rhCDKL5 protein released from the exosomes. All process of isolation, purification, filtration, centrifugation and lysis may be done at 4oC. The rhCDKL5 protein may be further purified from the supernatant using an affinity chromatography resin with or without buffer exchange may be further filtered and loaded onto a chromatography column, e.g., Ni-NTA column chromatography resin, using step elution at pH 7.5-8.0. The loaded chromatography column may be washed and eluted to obtain elution fractions which can be analyzed for presence of the purified rhCDKL5 protein linked to the exosome trafficking tag. Western blotting using an antibody specific to the CDKL5 protein, HIS tag or any other detectable tag or other suitable techniques may be used to detect the rhCDKL5 protein at any step of the process. Removal of Tag Peptide from Soluble, Non-Denatured rhCDKL5 Proteins

[0262] Following purification of the rhCDKL5 protein linked to at least one intracellular expression tag, at least one secretion signal tag, and / or an exosome trafficking tag, via a TEV cleavage site linker, as disclosed herein, the rhCDKL5 protein may be contacted with a TEV protease to cause TEV cleavage and subsequent removal of the tags. The method of removing the tags linked to the rhCDKL5 comprising a TEV cleavage site linker may comprise contacting the recombinant protein with about 1000 IU to about 1500 IU (e.g., about 1000 IU to about 1100 IU, about 1100 IU to about 1200 IU, about 1200 IU to about 1300 IU, about 1300 IU to about 1400 IU, about 1400 IU to about 1500 IU, about 1100 IU to about 1400 IU, about 1200 IU to about 1400 IU, about 1300 IU to about 1500 IU, and all integers between and including 1000 IU to 1500 IU) of the TEV protease per mg of total recombinant protein. The method of removing the tags linked to the rhCDKL5 via a TEV cleavage site linker maycomprise contacting the recombinant protein with the TEV protease at about 4oC to about 37oC (e.g., about 4oC to about 8oC, about 8oC to about 12oC, about 12oC to about 16oC, about 16oC to about 20oC, about 20oC to about 24oC, about 24oC to about 28oC, about 28oC to about 30oC, about 30oC to about 37oC, about 8oC to about 30oC, about 12oC to about 28oC, about 16oC to about 24oC, about 20oC to about 30oC, and all integers between and including about 4oC to about 37oC.

[0263] The method of removing the tags linked to the rhCDKL5 via a TEV cleavage site linker produced by the method disclosed herein, may comprise contacting the recombinant protein with the TEV protease at a pH of about 7.5-8.5 (e.g., about 7.5 to about 7.7, about 7.7 to about 8.0, about 8.0 to about 8.2, about 8.2 to about 8.5, about 7.7 to about 8.2, about 8.0 to about 8.5, and all integers between and including 7.5 to 8.5). The method of removing the tags linked to the rhCDKL5 via a TEV cleavage site linker produced by the method disclosed herein, may comprise contacting the recombinant protein with a TEV protease for about 1 hour to about 20 hours (e.g., about 1 hour to about 4 hours, about 4 hours to about 8 hours, about 8 hours to about 12 hours, about 12 hours to about 16 hours, about 16 hours to about 20 hours, about 4 hours to about 16 hours, about 8 hours to about 12 hours, and all integers between and including 1 hour and 20 hours). The method of removing the tags linked to the rhCDKL5 via a TEV cleavage site linker produced by the method disclosed herein, may comprise contacting the recombinant protein with a TEV protease in presence of a protein stabilizer selected from Arginine, Histidine, Proline, Lysine, Triton X-100 and Sorbitol or a combination thereof. The contacting of the rhCDKL5 protein with the TEV protease can be done in presence of a protein stabilizer selected from Arginine, Histidine, Proline, Lysine, Triton X-100 and Sorbitol, at about 100 to about 300 mM (e.g., about 100 mM to about 150 mM, about 150 mM to about 200 mM, about 200 mM to about 250 mM, about 250 mM to about 300 mM, about 150 mM to about 250 mM, about 200 mM to about 300 mM, about 100 mM to about 200 mM, and all integers between and including 100 mM and 300 mM).

[0264] The process of cleaving the TEV cleavage site linked tags from the rhCDKL5 protein may lead to formation of a post-reaction mixture comprising of a tagless CDKL5 protein, cleaved tag peptides (e.g., MBP or GFP) and partially digested tagged CDKL5 proteins (e.g., MBP-TEV-CDKL5 or GFP-TEV-CDKL5 proteins), as a result of an incomplete reaction. Post cleavage of the tags linked by a TEV cleavage site linker to the rhCDKL5 protein, the tagless CDKL5 protein can be further isolated from the post-reaction mixture by contacting themixture with an affinity chromatography resin that specifically binds to the tagged peptides and collecting the tagless CDKL5 protein in the flowthrough. The affinity chromatography resins can be any one of an anti-GFP antibody resin, an anti-MBP antibody resin, a dextrin sepharose resin, a Ni-NTA resin or a combination thereof.

[0265] Post cleavage of the tagged-CDKL5 protein, the fully cleaved CDKL5 protein can be purified from impurities (tag peptides and partially digested tagged CDKL5 proteins) by contacting the post-reaction mixture with an immobilized-metal affinity chromatography (IMAC) resin in presence of metal ions, in a binding buffer comprising a salt concentration of about 0.5M to about 1M (e.g., about 0.5M to about 0.6M, about 0.6M to about 0.7M, about 0.7M to about 0.8M, about 0.8M to about 0.9M, about 0.9M to about 1M, about 0.6M to about 0.9M, about 0.7M to about 0.9M, or about 0.5M to about 0.7M, and all integers between and including 0.5M and 1M), with a pH of about 8.0 to about 8.5 (e.g., about 8.0 to about 8.1, about 8.1 to about 8.2, about 8.2 to about 8.3, about 8.3 to about 8.4, about 8.4 to about 8.5, and all integers between and including 8.0 to 8.5). The salt can be any one of sodium chloride, sodium phosphate or magnesium chloride. High salt concentration increases protein solubility and allows the IMAC resin to specifically binds full-length tagless CDKL5 protein but not the impurities. The binding buffer can be any one of Tris-HCl based, ammonium phosphate based, or Tris-acetate based buffer. The tagless CDKL5 protein retained in the IMAC resin can be eluted using an elution buffer comprising a gradient of an agent that competes for either the resin or the bound protein. The elution buffer can comprise an increased concentration of imidazole up to 0.5 M (e.g., about 0.1M, about 0.2M, about 0.3M to about 0.4M, or about 0.5M), ammonium chloride up to about 0.15M (e.g., about 0.05M, about 0.06M, about 0.07M, about 0.08M, about 0.09M, about 0.1M or about 0.15M), or substances such as histamine or glycine with affinity for the chelated metal can be used. The elution buffer can have a pH of about 4.0 to about 6.0 (e.g., about 4.0 to about 4.5, about 4.5 to about 5.0, about 5.0 to about 5.5, about 5.5 to about 6.0, about 4.5 to about 5.5, about 5.0 to about 6.0, about 4.0 to about 5.0, all integers between and including 4.0 and 6.0).

[0266] Post IMAC mediated purification, the tagless CDKL5 protein can be further purified from any partially digested rhCDKL5 protein or other impurities by contacting with a hydrophobic interaction chromatography (HIC) resin. The HIC resin can be any one of a hydrophobic butyl, hydrophobic carboxy and hydrophobic methyl resin. The contacting with a HIC resin can be done in a binding buffer comprising any one of sodium chloride, potassiumchloride or ammonium sulphate. The binding buffer for contacting with the HIC resin can be any sodium chloride at progressively increasing concentrations from about 1M to about 2M (e.g., increasing from about 1M to about 1.2M, about 1.2M to about 1.5M, about 1.5M to about 1.7M, and about 1.7M to about 2M). The binding buffer for contacting with the HIC resin can be at a pH of about 5.0 to about 7.0 (e.g., about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, about 5.5 to about 6.5, about 6.0 to about 7.0, about 5.5 to about 6.5, and all integers between and including 5.0 and 7.0). For eluting the tagless CDKL5 protein bound to the HIC resin, the resin can be contacted with an elution buffer comprising the same salt as in the binding buffer but with progressively decreasing concentration. The elution buffer can comprise sodium chloride at progressively decreasing concentrations of about 2M to about 0M (e.g., decreasing from about 2M to about 1.7M, about 1.7M to about 1.5M, about 1.5M to about 1.2M, about 1.2M to about 1M. about 1M to 0.5M, and 0.5M to 0M). Uses of Soluble, Non-Denatured rhCDKL5 Proteins

[0267] The present disclosure provides methods for reliably producing rhCDKL5 proteins. The rhCDKL5 proteins produced according to the presently disclosed methods may be soluble or solubilized. The rhCDKL5 proteins produced according to the presently disclosed methods may be non-denatured. Accordingly, the rhCDKL5 proteins produced according to the presently disclosed methods can be used to characterize the rhCDKL5 proteins and / or to carry out in vitro analytical assays, for instance, where there is an interest in detecting presence of CDKL5 peptides and CDKL5-specific antibodies in samples. Various uses of the rhCDKL5 proteins produced according to the presently disclosed methods are further described below.

[0268] In some embodiments, the rhCDKL5 proteins produced according to the present disclosure are useful in analytical methods for detecting CDKL5-specific antibodies in a sample. For example, the rhCDKL5 proteins produced by the methods described herein can be used to detect CDKL5-specific anti-drug-antibodies (ADAs) in a sample.

[0269] ADAs may be raised by subject immune systems after receiving a particular drug, limiting effectiveness of the drug or limiting the patient’s tolerability of further doses of the drug. For viral vector-mediated gene therapies in particular, immunogenicity of the genetic material and / or the therapeutic gene product can give rise to humoral and cellular immune responses, including development of ADAs, in patients administered the gene therapy. Thispatient immune response can lead to loss of efficacy of the gene therapy over time and may impede the possibility of re-dosing subjects. The ability to evaluate immune responses and ADA formation in pre-clinical and clinical trials of rhCDKL5s is critical to show and improve therapeutic efficacy as well as manage patient immune responses to the therapies. However, the difficulties faced in reliably producing soluble, non-denatured rhCDKL5 protein have thwarted development of ADA detection assays for rhCDKL5 therapeutics.

[0270] Accordingly, the rhCDKL5s produced according to the present methods enable detection of ADAs raised in subjects administered a therapeutic CDKL5 gene or protein. The subjects may be human subjects or animals receiving rhCDKL5-based gene therapies.

[0271] Analytical methods for detecting ADAs may be based on colorimetric methods, enzyme immunoassays (EIA), radioimmunoassays (RIA), fluoroimmunoassays (FIA), chemiluminescent immunoassays (CLIA), or counting immunoassays (CIA).

[0272] Generally, analytical methods for detecting CDKL5-specific antibodies in a sample comprise immobilizing a rhCDKL5 protein produced by the method of the present disclosure or a fragment thereof to a solid surface, contacting the immobilized rhCDKL5 protein with the sample to bind the immobilized rhCDKL5 protein to the CDKL5-specific antibody (if present in the sample), forming a bipartite complex of the rhCDKL5 protein and the CDKL5-specific antibody, then contacting the bipartite complex with a detection reagent that binds specifically to the CDKL5-specific antibody and not the rhCDKL5 protein, and forming a tripartite complex of the detection reagent, the rhCDKL5 protein, and the CDKL5- specific antibody. In some embodiments, the detection reagent comprises a detectable moiety that generates a detectable signal, enabling detection of the signal to determine the presence of the CDKL5-specific antibodies in the sample.

[0273] Immobilizing the rhCDKL5 protein or fragment thereof may involve coupling the rhCDKL5 protein or fragment to a solid surface. For example, the rhCDKL5 protein or fragment may be immobilized via interaction with an anti-CDKL5 antibody which is crosslinked to the solid surface. Alternatively, the rhCDKL5 protein or fragment may be covalently crosslinked to the solid surface.

[0274] Exemplary solid surfaces for immobilizing a rhCDKL5 protein or fragment include polyethylene, polyacrylamide, polystyrene, agarose, glass, or silicone rubber substrates.

[0275] The CDKL5 protein to be immobilized to a solid surface can be a rhCDKL5 protein linked to one or more intracellular expression tag, one or more secretion signal tag or an exosome trafficking tag, produced by the method of the present disclosure. The rhCDKL5 protein to be immobilized to a solid surface can be further linked to a peptide tag that allows binding of the rhCDKL5 protein to a moiety that allows linking to the CDKL5 protein to a solid surface. The rhCDKL5 protein to be immobilized to a solid surface can be further linked to a biotinylable peptide tag at the N-terminus or the C-terminus of the rhCDKL5 protein, comprising an amino acid sequence according to SEQ ID NO: 14. The rhCDKL5 protein to be immobilized to a solid surface can be biotinylated at the lysine residue of the biotinylable peptide tag according to SEQ ID NO: 14. The biotinylated CDKL5 protein comprising the biotinylable peptide tag of the present disclosure can be linked to a streptavidin coated solid surface.

[0276] The rhCDKL5 protein to be immobilized to a solid surface can comprise the biotinylable peptide tag according to SEQ ID NO: 14 between any one of the one or more intracellular expression tag, one or more secretion signal tag or an exosome trafficking tag and the N-terminus of the CDKL5 protein. The rhCDKL5 protein to be immobilized to a solid surface can comprise the biotinylable peptide tag according to SEQ ID NO: 14 between any one of the one or more intracellular expression tag, one or more secretion signal tag or an exosome trafficking tag and the C-terminus of the CDKL5 protein. The rhCDKL5 protein to be immobilized to a solid surface can comprise the biotinylable peptide tag according to SEQ ID NO: 14 between an intracellular expression tag and the N-terminus of the CDKL5 protein. The rhCDKL5 protein to be immobilized to a solid surface can comprise the biotinylable peptide tag according to SEQ ID NO: 14 between an intracellular expression tag and the C- terminus of the CDKL5 protein.

[0277] The rhCDKL5 protein to be immobilized to a solid surface can comprise a biotinylable peptide tag according to SEQ ID NO: 14 between an intracellular expression tag, MBP-TEV and the N-terminus, and an intracellular expression tag, GFP-TEV linked to the C- terminus of the CDKL5 protein, with an amino acid sequence according to SEQ ID NO: 15. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 15. The rhCDKL5 protein to be immobilized to a solid surface can comprise anintracellular expression tag, MBP-TEV, linked to the N-terminus, and a biotinylable peptide tag according to SEQ ID NO: 14 between an intracellular expression tag, GFP-TEV and the C- terminus of the CDKL5 protein, with an amino acid sequence according to SEQ ID NO: 16. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 16.

[0278] The rhCDKL5 protein to be immobilized to a solid surface can comprise a biotinylable peptide tag according to SEQ ID NO: 14 between an intracellular expression tag, GFP-TEV and the N-terminus, and an intracellular expression tag, MBP-TEV linked to the C- terminus of the CDKL5 protein, with an amino acid sequence according to SEQ ID NO: 17. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 17. The rhCDKL5 protein to be immobilized to a solid surface can comprise an intracellular expression tag, GFP-TEV, linked to the N-terminus, and a biotinylable peptide tag according to SEQ ID NO: 14 between an intracellular expression tag, MBP-TEV and the C- terminus of the CDKL5 protein, with an amino acid sequence according to SEQ ID NO: 18. In certain embodiments, the rhCDKL5 protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 18.

[0279] Analytical methods for detecting CDKL5-specific antibodies in a sample comprise immobilizing a rhCDKL5 protein or a fragment thereof may also comprise additional steps to reduce non-specific interactions. For example, the methods may comprise contacting the immobilized rhCDKL5 protein with a coating buffer or a blocker before proceeding with applying the detection reagent. The methods may also comprise, e.g., a step of contacting the bipartite complex with a wash buffer before proceeding with applying the detection reagent. Once the detection reagent is added, an additional wash step using a wash buffer can be performed before proceeding to detect a signal from the detection reagent. The detection reagent can be an antibody or a fragment thereof that can specifically bind CDKL5 specific antibody.

[0280] Detection reagents useful in the present methods may comprise, e.g., a detectable moiety selected from an enzyme, a fluorophore, or a radioisotope. The detectable signal from the detectably moiety may be, e.g., a colorimetric signal, a fluorescent signal, or radiation. In some embodiments, the detectable moiety is an enzyme, and detecting the signal comprises contacting the tripartite complex with a substrate for generating the detectable signal.

[0281] Examples of the enzyme include a horse radish peroxidase, an alkaline phosphatase, a galactosidase, an acetylcholinesterase, or a catalase. Examples of fluorophore- based detectable moieties include fluorescein isothiocyanate (FITC), phycoerythrin (PE) or a conjugate thereof, Alexa Fluor®dyes, rhodamine dyes, Cy-based dyes, orange G-dyes, Texas red, and the like. Examples of radioisotope-based detectable moieties include90Y,111In,177Lu,99mTc,123I,125I and131I.

[0282] The detection reagent can be a rhCDKL5 protein produced by the method of the present disclosure. The detectable reagent that is a rhCDKL5 protein produced by the method of the present disclosure comprises a partner 1 peptide tag, which can bind irreversibly to a partner 2 protein that is labelled with a detectable moiety. Direct labelling or linking of a CDKL5 protein by a detectable moiety can lead to misfolding and masking of epitopes on the protein surface due to the presence of the intrinsically disordered domains. The detectable moiety can be a luminescent moiety selected from any one of AlexaFluor647, any one of Ruthenium (Ru), fluorescein isothiocyanate (FITC), phycoerythrin (PE) or a conjugate thereof, an Alexa Fluor®dye, a rhodamine dye, a Cy-based dye, an Orange- G-dye, or Texas red. The detectable moiety can be a luminescent moiety selected from any one of the partner 1 peptide tag and the partner 2 protein can be labelling and conjugating SpyTag / SpyCatcher systems known in the art, including but not limited to BioRad SpyCatcher and SpyTag products, Kerafast Biotech SpyTag / SpyCatcher Protein Coupling Reagents or Addgene SpyTag-β- Lactamase-SpyCatcher proteins.

[0283] As an example, the methods described herein of detecting CDKL5 specific antibody present in a sample may comprise: contacting the (a) sample solution and (b) each dilution of standard solution comprising known amount of a CDKL5 specific antibody, with the capture reagent that is a biotinylated rhCDKL5 protein of the present disclosure comprising a biotinylable peptide tag sequence immobilized to a streptavidin coated solid surface and a CDKL5 detection reagent of the present disclosure that comprises a partner 1 peptide tag boundto a partner 2 protein linked to detectable moiety, and detecting a sample signal from the tripartite complex formed between the biotinylated rhCDKL5 protein, CDKL5 specific antibody, and the CDKL5 detection reagent in the sample solution in the sample and each dilution of standard solution; measuring the sample signal generated from the tripartite complex formed in the sample and the standard solution; and comparing the signals from the CDKL5 detection reagent in the sample solution and each dilution of the standard solution to determine the concentration of CDKL5 present in the sample.

[0284] Detection reagents in the method for quantitatively measuring an amount of CDKL5 protein in a sample disclosed herein can be any detection reagent that specifically binds to a rhCDKL5 protein, such as an antibody or a fragment thereof.

[0285] In another aspect, the rhCDKL5 proteins produced by the methods described herein, or fragments thereof, can be used to prepare a standard solution for use in a method of quantitatively measuring an amount of CDKL5 present in a sample solution. Measuring an amount of a CDKL5 protein present in a sample is powerful way to evaluate potency of a candidate rhCDKL5 therapeutic. For example, clinical and preclinical subjects may be administered a gene therapy product, but the amount of CDKL5 protein expressed in cells of the subject may vary. Confirming that the therapeutic protein is expressed at levels sufficient to maintain a therapeutic effect is an important aspect of validating any candidate therapy. However, validating an analytical method for quantitatively measuring CDKL5 protein present in a sample, such as a sample from a subject administered a rhCDKL5 therapeutic, requires comparison against a standard with a known amount of rhCDKL5 protein presence. Previous inability to reliably produce soluble, non-denatured rhCDKL5 proteins have made quantitative evaluation of CDKL5 proteins present in samples collected from subject difficult. Thus, the methods provided herein can produce rhCDKL5 protein for use in methods of quantitatively measuring the amount of CDKL5 present in a sample.

[0286] In some embodiments, the method of quantitatively measuring an amount of CDKL5 present in a sample solution entails a colorimetric method, an enzyme immunoassay (EIA), a radioimmunoassay (RIA), a fluoroimmunoassay (FIA), a chemiluminescent immunoassay (CLIA), or a counting immunoassay (CIA).

[0287] The method of quantitatively measuring an amount of CDKL5 present in a sample solution may comprise, e.g., contacting the sample solution with a CDKL5 capturereagent and a CDKL5 detection reagent, and detecting a signal from the CDKL5 detection reagent in the sample solution. A signal from the CDKL5 detection reagent can be measured and then compared to measurements taken in the same manner from one or more standard solutions comprising known amounts of the rhCDKL5 protein.

[0288] For example, dilutions of the standard solution comprising the rhCDKL5 protein may be contacted with a CDKL5 capture reagent and a CDKL5 detection reagent, and a signal from the CDKL5 detection reagent in each dilution of the standard solution may be detected. In this way, a standard calibration curve can be generated using the signal from the CDKL5 detection reagent at each dilution of the standard solution. Comparing the CDKL5 detection reagent signal with the calibration curve, one can quantitatively determine the concentration of CDKL5 in the sample solution. Dilutions of the standard solution can be made to generate the calibration curve for comparing the CDKL5 detection reagent signal with the calibration curve, and quantitatively determining the concentration of CDKL5 in the sample. In some embodiments, serial dilutions of a standard solution containing a known concentration of the rhCDKL5 protein produced according to the methods described herein are generated to prepare the standard calibration curve.

[0289] In certain embodiments, the CDKL5 capture reagent binds specifically to CDKL5 in the sample solution and the rhCDKL5 protein or a fragment thereof in the standard solution. The CDKL5 capture reagent may be an antibody or a fragment thereof that specifically binds to CDKL5 protein. The CDKL5 capture reagent may be immobilized, for example, to a solid surface, for use in the quantitative analytical methods described herein. For instance, the CDKL5 capture reagent may be immobilized by any one of protein A, protein G or Protein L to a solid surface.

[0290] In some embodiments, the CDKL5 detection reagent, on the other hand, is capable of binding specifically to CDKL5 in the sample solution and the CDKL5 protein or a fragment thereof in the standard solution but not to the CDKL5 capture reagent. That is, the CDKL5 detection reagent will not cross-react with the CDKL5 capture reagent or the solid surface to which the CDKL5 capture reagent is immobilized. The CDKL5 detection reagent can be, e.g., an antibody. In some embodiments, CDKL5 capture reagent is an antibody that is different from the CDKL5 capture reagent, and may be used at a concentration, e.g., between about 0.5 mg / ml to about 2 mg / ml (e.g., 0.5 mg / ml to 0.75 mg / ml, 0.75 mg / ml to 1 mg / ml, 1 mg / ml to 1.25 mg / ml, 1.25 mg / ml to 1.5 mg / ml, 1.5 mg / ml to 1.75 mg / ml, 1.75 mg / ml to 2.0mg / ml, 0.75 mg / ml to 1.75 mg / ml, 1 mg / ml to 1.5 mg / ml, and all integers between and including 0.5 mg / ml and 2 mg / ml).

[0291] In some embodiments, detecting a signal from the CDKL5 detection reagent may comprise contacting the detection reagent with a secondary detection reagent that specifically binds the CDKL5 detection reagent and not the CDKL5 capture reagent. The secondary detection can be, e.g., an antibody or a fragment thereof and may optionally further comprise a reporter moiety. Examples of suitable reporter moieties are known. The reporter moiety can be a detectable moiety that can be any one of an enzyme, a fluorophore, or a radioisotope. The reporter moiety can be any one of a horse radish peroxidase, an alkaline phosphatase, a galactosidase, an acetylcholinesterase, or a catalase. The reporter moiety can be any one of fluorescein isothiocyanate (FITC), phycoerythrin (PE) or a conjugate thereof, an Alexa Fluor®dye, a rhodamine dye, a Cy-based dye, a G-dye, or Texas red. The reporter moiety can be any one of90Y,111In,177Lu,99mTc,123I,125I, or131I.

[0292] As an example, the methods described herein of quantitatively measuring CDKL5 present in a sample may comprise: contacting the sample and dilutions of a standard solution created with known amounts of rhCDKL5 protein with a CDKL5 capture reagent and a CDKL5 detection reagent in a coating or blocking buffer; detecting a signal from the CDKL5 detection reagent in the sample solution and each dilution of the standard solution (e.g., by contacting the CDKL5 detection reagent with a detection solution); and comparing the signals from the CDKL5 detection reagent in the sample solution and each dilution of the standard solution to determine the concentration of CDKL5 present in the sample.

[0293] The sample or sample solution may be, e.g., from one or more subjects suffering from or suspected of having a CDKL5 deficiency disorder (CDD), CDKL5 encephalopathy, CDKL5-related epilepsy, CDKL5-related epileptic encephalopathy, early infantile epileptic encephalopathy 2. In some embodiments, the samples or sample solutions are from subject(s) to whom a therapeutic rhCDKL5 gene or protein has been administered or is to be administered. Kits for Detecting and Quantitatively Measuring CDKL5 Proteins and CDKL5 Protein Specific Antibody

[0294] Development of treatment and therapeutics for disorders related to deficiency of CDKL5 requires efficient method and reagents for detection and diagnosis of the presenceor absence of the CDKL5 protein in samples from patients and experimental animals before and after administration of the treatment.

[0295] The present disclosure provides a kit for detecting CDKL5 specific antibody in a sample, comprising: a recombinant human CDKL5 (rhCDKL5) protein or a fragment thereof, produced by the method of the present disclosure; and a detection reagent that specifically binds to the CDKL5 specific antibody but not to the rhCDKL5 protein or fragment thereof, wherein the detection reagent comprises a detectable moiety.

[0296] The kit for detecting CDKL5 specific antibody in a sample can further comprise i. a solid surface; ii. a coating / blocking solution; iii. a washing solution; iv. a detection solution; or v. a combination thereof. The solid surface can be any one of polyethylene, polyacrylamide, polystyrene, agarose, glass, or silicone rubber. The solid surface can be coated with streptavidin for binding to and immobilizing the biotinylated rhCDKL5 protein produced by the method of the present disclosure.

[0297] The rhCDKL5 protein of the kit for detecting CDKL5 specific antibody in a sample, can be linked to one or more intracellular expression tag, one or more secretion signal tag or an exosome trafficking tag, produced by the method of the present disclosure. The rhCDKL5 protein of the kit for detecting CDKL5 specific antibody in a sample, is immobilized to a solid surface.

[0298] The rhCDKL5 protein to be immobilized to a solid surface can be further linked to a peptide tag that allows binding of the rhCDKL5 protein to a moiety that allows linking to the CDKL5 protein to a solid surface. The rhCDKL5 protein can be further linked to a biotinylable peptide tag at the N-terminus or the C-terminus of the rhCDKL5 protein, comprising an amino acid sequence according to SEQ ID NO: 14. The rhCDKL5 protein can be biotinylated at the lysine residue of the biotinylable peptide tag according to SEQ ID NO: 14. The rhCDKL5 protein can comprise the biotinylable peptide tag according to SEQ ID NO: 14 between any one of the one or more intracellular expression tag, one or more secretion signal tag or an exosome trafficking tag and the N-terminus of the CDKL5 protein. The rhCDKL5 protein can comprise the biotinylable peptide tag according to SEQ ID NO: 14 between any one of the one or more intracellular expression tag, one or more secretion signal tag or an exosome trafficking tag and the C-terminus of the CDKL5 protein. The rhCDKL5 protein can comprise the biotinylable peptide tag according to SEQ ID NO: 14 between an intracellularexpression tag and the N-terminus of the CDKL5 protein. The rhCDKL5 protein can comprise the biotinylable peptide tag according to SEQ ID NO: 14 between an intracellular expression tag and the C-terminus of the CDKL5 protein.

[0299] The rhCDKL5 protein produced by the method of the present disclosure can comprise a biotinylable peptide tag according to SEQ ID NO: 14 between an intracellular expression tag, MBP-TEV and the N-terminus, and an intracellular expression tag, GFP-TEV linked to the C-terminus of the CDKL5 protein, with an amino acid sequence according to SEQ ID NO: 15 (or an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 15). The rhCDKL5 protein produced by the method of the present disclosure can comprise an intracellular expression tag, MBP-TEV, linked to the N-terminus, and a biotinylable peptide tag according to SEQ ID NO: 14 between an intracellular expression tag, GFP-TEV and the C-terminus of the CDKL5 protein, with an amino acid sequence according to SEQ ID NO: 16 (or an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 16).

[0300] The rhCDKL5 protein produced by the method of the present disclosure can comprise a biotinylable peptide tag according to SEQ ID NO: 14 between an intracellular expression tag, GFP-TEV and the N-terminus, and an intracellular expression tag, MBP-TEV linked to the C-terminus of the CDKL5 protein, with an amino acid sequence according to SEQ ID NO: 17 (or an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 17). The rhCDKL5 protein produced by the method of the present disclosure can comprise an intracellular expression tag, GFP-TEV, linked to the N-terminus, and a biotinylable peptide tag according to SEQ ID NO: 14 between an intracellular expression tag, MBP-TEV and the C-terminus of the CDKL5 protein, with an amino acid sequence according to SEQ ID NO: 18 (or an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 18).

[0301] The kit for detecting CDKL5 specific antibody in a sample of the present disclosure can further comprise one or more biotinylation reagents selected from Biotin acid (e.g., Biotin-PEG-acid), Biotin-NHS ester (e.g., Biotin-PEG-NHS ester, Sulfo-NHS-Biotin or Destiobiotin-NHS-ester), Biotin-EDA-NHS, NHS-PEG-biotinidase resistant biotin, Biotin- PFP, Biotin-EDA-PFP, Biotin-TFP, TFP-PEG-biotinidase resistant biotin, N-Bpc-Biocytin and PEG-Biotin-PEG, for crosslinking biotin to the lysine residue of the biotinylable peptide tag.

[0302] The kit for detecting CDKL5 specific antibody in a sample of the present disclosure can further comprise an anti-CDKL5 antibody or a crosslinker for immobilizing the rhCDKL5 protein or fragment thereof to the solid surface. The crosslinker can comprise any one of maleimide, sulfhydryl reactive groups, or succinimidyl esters. The solid surface can be coated with protein A, protein G or protein L to bind and immobilize the anti-CDKL5 antibody on the solid surface. The solid surface can comprise the anti-CDKL5 antibody immobilized by protein A.

[0303] The detection moiety of the detection reagent of the kit for detecting CDKL5 specific antibody in a sample of the present disclosure can be any one of an enzyme, a fluorophore, or a radioisotope. The detectable moiety can generate a detectable signal which is any one of a colorimetric signal (if the detection moiety is an enzyme), a fluorescent signal (if the detection moiety is a fluorophore), or radiation (if the detection moiety is an radioisotope). The enzyme can be any one of a horse radish peroxidase, an alkaline phosphatase, a galactosidase, an acetylcholinesterase, or a catalase. If the detectable moiety is an enzyme, then the kit for detecting CDKL5 specific antibody in a sample of the present disclosure can further comprise a substrate that generates a detectable signal upon reacting with the enzyme. The substrate can be any one of 3,3',5,5'-tetramethylbenzidine (TMB) and 2,2' -azino-di-[3- ethylbenzthiazoline-6-sulfonic acid] (ABTS) (if the enzyme is a horse radish peroxidase), p- nitrophenyl phosphate (if the enzyme is an alkaline phosphatase), Bluo-gal and X-gal (if the enzyme is a galactosidase), Acetylthiocholine (ATCh) (if the enzyme is an acetylcholinesterase) or hydrogen peroxide (if the enzyme is a catalase).

[0304] The detectable moiety can be a fluorophore selected from any one of fluorescein isothiocyanate (FITC), phycoerythrin (PE) or a conjugate thereof, an Alexa Fluor® dye, a rhodamine dye, a Cy-based dye, an Orange G-dye, or Texas red. The detectable moiety can bea radioisotope selected from any one of90Y,111In,177Lu,99mTc123I,125I, or131I. The detection reagent can be an antibody or a fragment thereof.

[0305] The detection reagent of the kit for detecting CDKL5 specific antibody in a sample of the present disclosure can be a rhCDKL5 protein produced by the method of the present disclosure. The rhCDKL5 protein detectable reagent can be linked to a partner 1 peptide tag, which can bind irreversibly to a partner 2 protein that is labeled with a detectable moiety. Direct labeling or linking of a CDKL5 protein by a detectable moiety can lead to misfolding and masking of epitopes on the protein surface due to the presence of the intrinsically disordered domains. The detectable moiety can be a luminescent moiety selected from any one of AlexaFluor647, any one of Ruthenium (Ru), fluorescein isothiocyanate (FITC), phycoerythrin (PE) or a conjugate thereof, an Alexa Fluor®dye, a rhodamine dye, a Cy-based dye, a G-dye, or Texas red. The detectable moiety can be a luminescent moiety selected from any one of the partner 1 peptide tag and the partner 2 protein can be labeling and conjugating SpyTag / SpyCatcher systems known in the art, including but not limited to BioRad SpyCatcher and SpyTag products, Kerafast Biotech SpyTag / SpyCatcher Protein Coupling Reagents or Addgene SpyTag-β-Lactamase-SpyCatcher proteins.

[0306] The present disclosure also provides a kit for quantitatively measuring an amount of CDKL5 present in a sample solution comprising: i. a CDKL5 capture reagent; ii. a CDKL5 detection reagent; and iii. a standard solution comprising a recombinant human CDKL5 (rhCDKL5) protein produced by the method of the present disclosure or a fragment thereof, or a lyophilized reagent for preparing said standard solution. The CDKL5 detection reagent binds specifically to CDKL5 in the sample and the rhCDKL5 protein or a fragment thereof in the standard solution. The CDKL5 protein can be a human CDKL5 protein or a fragment thereof.

[0307] The CDKL5 detection reagent of the kit for quantitatively measuring an amount of CDKL5 present in a sample solution can bind specifically to CDKL5 in the sample and the rhCDKL5 protein or a fragment thereof in the standard solution. The CDKL5 detection reagent can be an antibody or a fragment thereof that specifically binds to CDKL5 protein.

[0308] The kit for quantitatively measuring an amount of CDKL5 present in a sample solution can further comprise i. a solid surface; ii. a coating solution; iii. a washing solution;iv. a detection solution; or v. a combination thereof. The solid surface can be any one of polyethylene, polyacrylamide, polystyrene, agarose, glass, or silicone rubber.

[0309] The CDKL5 capture reagent of the kit for quantitatively measuring an amount of CDKL5 present in a sample solution can bind specifically to CDKL5 in the sample and the rhCDKL5 protein or a fragment thereof in the standard solution. The CDKL5 detection reagent can be a human CDKL5 specific antibody or a fragment thereof, that specifically binds to CDKL5 at a site that is different from the binding site of the CDKL5 detection reagent. The CDKl5 capture reagent can be immobilized to the solid surface. the CDKL5 capture reagent can be immobilized by protein A, protein G or protein L to the solid surface. The CDKL5 capture reagent can be immobilized to the solid surface. The CDKL5 capture reagent can be immobilized by protein A to the solid surface.

[0310] The CDKL5 capture reagent of the kit for quantitatively measuring an amount of CDKL5 present in a sample solution can bind specifically to CDKL5 in the sample and the rhCDKL5 protein or a fragment thereof in the standard solution but does not bind to the CDKL5 capture reagent. The CDKL5 detection reagent is different from the CDKL5 capture reagent.

[0311] The CDKL5 capture reagent of the kit for quantitatively measuring an amount of CDKL5 present in a sample solution, can be directly labelled with a detectable moiety. The detectable moiety can be any one of any one of an enzyme, a fluorophore, or a radioisotope. The enzyme can be any one of a horse radish peroxidase, an alkaline phosphatase, a galactosidase, an acetylcholinesterase, or a catalase. The fluorophore can be any one of fluorescein isothiocyanate (FITC), phycoerythrin (PE) or a conjugate thereof, an Alexa Fluor®dye, a rhodamine dye, a Cy-based dye, a G-dye, or Texas red. The radioisotope can be any one of90Y,111In,177Lu,99mTc123I,125I, or131I.

[0312] The kit for quantitatively measuring an amount of CDKL5 present in a sample solution, can further comprise a secondary detection reagent that specifically binds the CDKL5 detection reagent and not the CDKL5 capture reagent. The secondary detection reagent can be an antibody or a fragment thereof. The secondary detection reagent can be an antibody or a fragment thereof, can comprise a reporter moiety. The reporter moiety can be a detectable moiety that can be any one of an enzyme, a fluorophore, or a radioisotope. The reporter moiety can be any one of a horse radish peroxidase, an alkaline phosphatase, a galactosidase, an acetylcholinesterase, or a catalase. The reporter moiety can be any one of fluoresceinisothiocyanate (FITC), phycoerythrin (PE) or a conjugate thereof, an Alexa Fluor®dye, a rhodamine dye, a Cy-based dye, a G-dye, or Texas red. The reporter moiety can be any one of90Y,111In,177Lu,99mTc123I,125I, or131I. Method of expressing and detecting CDKL5 protein of present disclosure in a subject

[0313] Development of effective treatment and therapeutic strategies for CDKL5 deficiency disorder (CDD) requires introducing or expressing a human CDKL5 protein into brain tissue of subjects to replace deficient CDKL5 protein production. An important aspect of a method of expressing or introducing a human CDKL5 protein in a tissue of a subject is the ability to determine the presence and / or level of the protein in the tissue of the subject. The present disclosure also provides methods of specifically detecting human CDKL5 protein in samples obtained from the subject expressing the human CDKL5 protein. The method of the present disclosure can be used for specifically detecting any introduced or expressed human CDKL5 protein from endogenous CDKL5 of a subject that is a non-human experimental animal, for example, a rodent, a non-human primate or a canine. Detecting a human CDKl5 protein in tissue samples of a subject, especially a non-human primate is particularly difficult in view of (1) the low abundance of the target protein, and (2) targeting a single amino acid difference between the human and monkey CDKL5.

[0314] The present disclosure provides a method of expressing CDKL5 protein in a subject in need thereof, by administering to the subject a therapeutically effective amount of a human CDKL5 protein generated by the method of the present disclosure. The subject can be a human, a non-human primate, a rodent or a canine. The administering of a therapeutically effective amount of the human CDKL5 protein can be done via an intrathecal (IT), intra cisterna magna (ICM) or an intracerebroventricular (ICV) route.

[0315] The method of expressing CDKL5 protein in a subject of the present disclosure can further comprise, detecting the presence of human CDKL5 in the subject by: i) providing a sample from the subject; ii) contacting the sample with one or more proteolytic enzyme(s) for an amount of time to generate peptide fragments; iii) detecting the presence of a human specific CDKL5 sequence in the peptide fragments of step ii). The method of expressing CDKL5 protein can comprise contacting the sample with one or more proteolytic enzyme(s) selected from a group consisting of trypsin, chymotrypsin, Lys-C, AspN, GluC, chymotrypsin,and proteinase K. The method can comprise contacting the sample with Lys-C and chymotrypsin.

[0316] In some embodiments, the method comprises detecting of the presence of a human specific CDKL5 sequence in the peptide fragments by any one of a mass spectrometry (MS), colorimetric method, enzyme immunoassay (EIA), radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLIA), or counting immunoassay (CIA). In some embodiments, the mass spectrometry is a liquid chromatography- mass spectrometry (LC-MS). In some embodiments, the LC-MS comprises any one of adsorption chromatography, partition chromatography, ion-exchange chromatography, size- exclusion chromatography, and affinity chromatography. In some embodiments, the human specific CDKL5 peptide comprises an amino acid sequence according to SEQ ID NO: 28 (ETSREGTSSF).

[0317] For example, detecting of the human CDKL5 protein in subject, for e.g., a non- human primate subject, can be done by isolating the brain tissue for the subject followed by homogenization of the tissue and isolation of intracellular proteins. The intracellular proteins can be further purified from the homogenized tissue by an intermediate step of contacting with ion exchange chromatography resin, for e.g., a cation exchange chromatography resin, a hydrophobic interaction chromatography resin, a mix mode chromatography resin or a combination thereof. The intracellular proteins obtained from the homogenized tissue can be digested or cleaved into smaller peptides by contacting with a combination of chymotrypsin and Lys-C. The digested proteins can be contacted with a liquid chromatography resin and the flow through can be analyzed by mass spectrometry to detect a human specific CDKL5 peptide, ETSREGTSSF (SEQ ID NO: 28), wherein presence of the peptide will indicate expression and / or efficient replacement of the human CDKL5 protein in the subject. CDKL5 specific monoclonal antibodies

[0318] The present disclosure also provides an anti-CDKL5 antibody or a fragment thereof. In some embodiments, the anti-CDKL5 antibody or fragment thereof binds the non- catalytic / non-kinase domain of a human CDKL5 protein. In some embodiments, the anti- CDKL5 antibody or fragment thereof binds the catalytic / kinase domain of a human CDKL5 protein.

[0319] In some embodiments, the antibody fragments described herein can be any one of an antibody heavy chain, an antibody light chain, a single chain antibody, a single-domain antibody (for example, a variable heavy domain of heavy chain (VHH)), Fab antibody fragment, Fv antibody fragment, diabody, F(ab′)2 antibody fragment, Fab′ antibody fragment and single-chain Fv (scFv) antibody fragment.

[0320] In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises any combination of heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3), as described in Table 2. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises any combination of light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3), as described in Table 2. In some embodiments, the anti-CDKL5 antibody or fragment thereof comprises any of the combination of variable heavy chain and variable light chain sequences, as described in Table 2.

[0321] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain complementarity determining region 1 (HCDR1) according to SEQ ID NO: 42, a HCDR2 according to SEQ ID NO: 43 and a HCDR3 according to SEQ ID NO: 44.

[0322] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain complementarity determining region 1 (HCDR1) according to SEQ ID NO: 45, a HCDR2 according to SEQ ID NO: 46 and a HCDR3 according to SEQ ID NO: 47.

[0323] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain complementarity determining region 1 (HCDR1) according to SEQ ID NO: 48, a HCDR2 according to SEQ ID NO: 49 and a HCDR3 according to SEQ ID NO: 50.

[0324] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain complementarity determining region 1 (HCDR1) according to SEQ ID NO: 51, a HCDR2 according to SEQ ID NO: 52 and a HCDR3 according to SEQ ID NO: 53.

[0325] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain complementarity determining region 1 (HCDR1) according to SEQID NO: 54, a HCDR2 according to SEQ ID NO: 55 and a HCDR3 according to SEQ ID NO: 56.

[0326] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain complementarity determining region 1 (HCDR1) according to SEQ ID NO: 57, a HCDR2 according to SEQ ID NO: 58 and a HCDR3 according to SEQ ID NO: 59.

[0327] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain complementarity determining region 1 (LCDR1) according to SEQ ID NO: 60, a LCDR2 according to LVS and a LCDR3 according to SEQ ID NO: 62.

[0328] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain complementarity determining region 1 (LCDR1) according to SEQ ID NO: 63, a LCDR2 according to KVS and a LCDR3 according to SEQ ID NO: 65.

[0329] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain complementarity determining region 1 (LCDR1) according to SEQ ID NO: 66, a LCDR2 according to KVS and a LCDR3 according to SEQ ID NO: 67.

[0330] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain complementarity determining region 1 (LCDR1) according to SEQ ID NO: 68, a LCDR2 according to LVS and a LCDR3 according to SEQ ID NO: 69.

[0331] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain complementarity determining region 1 (LCDR1) according to SEQ ID NO: 70, a LCDR2 according to WAS and a LCDR3 according to SEQ ID NO: 72.

[0332] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain complementarity determining region 1 (LCDR1) according to SEQ ID NO: 73, a LCDR2 according to QVS and a LCDR3 according to SEQ ID NO: 75.

[0333] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain complementarity determining region 1 (HCDR1) according to SEQ ID NO: 42, a HCDR2 according to SEQ ID NO: 43, a HCDR3 according to SEQ ID NO: 44, a light chain complementarity determining region 1 (LCDR1) according to SEQ ID NO: 60, a LCDR2 according to LVS and a LCDR3 according to SEQ ID NO: 62.

[0334] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain complementarity determining region 1 (HCDR1) according to SEQ ID NO: 45, a HCDR2 according to SEQ ID NO: 46, a HCDR3 according to SEQ ID NO: 47, a light chain complementarity determining region 1 (LCDR1) according to SEQ ID NO: 63, a LCDR2 according to KVS and a LCDR3 according to SEQ ID NO: 65.

[0335] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain complementarity determining region 1 (HCDR1) according to SEQ ID NO: 48, a HCDR2 according to SEQ ID NO: 49, a HCDR3 according to SEQ ID NO: 50, a light chain complementarity determining region 1 (LCDR1) according to SEQ ID NO: 66, a LCDR2 according to KVS and a LCDR3 according to SEQ ID NO: 67.

[0336] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain complementarity determining region 1 (HCDR1) according to SEQ ID NO: 51, a HCDR2 according to SEQ ID NO: 52, a HCDR3 according to SEQ ID NO: 53, a light chain complementarity determining region 1 (LCDR1) according to SEQ ID NO: 68, a LCDR2 according to LVS and a LCDR3 according to SEQ ID NO: 69.

[0337] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain complementarity determining region 1 (HCDR1) according to SEQ ID NO: 54, a HCDR2 according to SEQ ID NO: 55, a HCDR3 according to SEQ ID NO: 56, a light chain complementarity determining region 1 (LCDR1) according to SEQ ID NO: 70, a LCDR2 according to WAS and a LCDR3 according to SEQ ID NO: 72.

[0338] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain complementarity determining region 1 (HCDR1) according to SEQ ID NO: 57, a HCDR2 according to SEQ ID NO: 58, a HCDR3 according to SEQ ID NO: 59, a light chain complementarity determining region 1 (LCDR1) according to SEQ ID NO: 73, a LCDR2 according to QVS and a LCDR3 according to SEQ ID NO: 75.

[0339] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 76. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain comprising an amino acid sequence that is at least 95% identical to anamino acid sequence according to SEQ ID NO: 76. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain according to SEQ ID NO: 76. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 77. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 77. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain according to SEQ ID NO: 77. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 78. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 78. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain according to SEQ ID NO: 78. In some embodiments, the anti- CDKL5 antibody or a fragment thereof comprises a variable heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 79. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 79. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain according to SEQ ID NO: 79. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 80. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 80. In some embodiments, theanti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain according to SEQ ID NO: 80. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 81. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 81. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain according to SEQ ID NO: 81.

[0340] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 82. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable light chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 82. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable light chain according to SEQ ID NO: 82. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 83. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable light chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 83. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable light chain according to SEQ ID NO: 83. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 84. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable light chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 84. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variablelight chain according to SEQ ID NO: 84. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 85. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable light chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 85. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable light chain according to SEQ ID NO: 85. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 86. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable light chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 86. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable light chain according to SEQ ID NO: 86. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 87. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable light chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 87. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable light chain according to SEQ ID NO: 87.

[0341] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain according to SEQ ID NO: 76 and a variable light chain according to SEQ ID NO: 82. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain according to SEQ ID NO: 77 and a variable light chain according to SEQ ID NO: 83. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain according to SEQ ID NO: 78 and a variable light chain according to SEQ ID NO: 84. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain according to SEQ ID NO: 79 and avariable light chain according to SEQ ID NO: 85. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain according to SEQ ID NO: 80 and a variable light chain according to SEQ ID NO: 86. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a variable heavy chain according to SEQ ID NO: 81 and a variable light chain according to SEQ ID NO: 87.

[0342] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 88. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 88. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain according to SEQ ID NO: 88. In some embodiments, the anti- CDKL5 antibody or a fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 89. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 89. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain according to SEQ ID NO: 89. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 90. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 90. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain according to SEQ ID NO: 90. In some embodiments, the anti- CDKL5 antibody or a fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 91. In some embodiments, the anti-CDKL5 antibody or afragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 91. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain according to SEQ ID NO: 91. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 92. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 92. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain according to SEQ ID NO: 92. In some embodiments, the anti- CDKL5 antibody or a fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 93. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 93. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain according to SEQ ID NO: 93.

[0343] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 94. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 94. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain according to SEQ ID NO: 94. In some embodiments, the anti- CDKL5 antibody or a fragment thereof comprises a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 95. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain comprising an amino acid sequence that is at least95% identical to an amino acid sequence according to SEQ ID NO: 95. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain according to SEQ ID NO: 95. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 96. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 96. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain according to SEQ ID NO: 96. In some embodiments, the anti- CDKL5 antibody or a fragment thereof comprises a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 97. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 97. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain according to SEQ ID NO: 97. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 98. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 98. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain according to SEQ ID NO: 98. In some embodiments, the anti- CDKL5 antibody or a fragment thereof comprises a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence according to SEQ ID NO: 99. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a light chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID NO: 99. In some embodiments,the anti-CDKL5 antibody or a fragment thereof comprises a light chain according to SEQ ID NO: 99.

[0344] In some embodiments, the anti-CDKL5 antibody or fragment thereof, comprises a variable heavy chain region according to any one of SEQ ID NOs.: 76-81, and a heavy chain constant region of mouse IgG1, mouse IgG2a, mouse IgG2b or mouse IgG3 isotype. In some embodiments, the anti-CDKL5 antibody or fragment thereof, comprises a variable heavy chain region according to any one of SEQ ID NOs.: 76-81, and a heavy chain constant region of mouse IgG1. In some embodiments, the anti-CDKL5 antibody or fragment thereof, comprises a variable heavy chain region according to any one of SEQ ID NOs.: 76-81, and a heavy chain constant region of mouse IgG2b. In some embodiments, the anti-CDKL5 antibody or fragment thereof, comprises a variable heavy chain region according to any one of SEQ ID NOs.: 76-81, and a heavy chain constant region of mouse IgG2a. In some embodiments, the anti-CDKL5 antibody or fragment thereof, comprises a variable heavy chain region according to any one of SEQ ID NOs.: 76-81, and a heavy chain constant region of mouse IgG3.

[0345] In some embodiments, the anti-CDKL5 antibody or fragment thereof, comprises a variable light chain region according to any one of SEQ ID NOs.: 82-87, and a light chain constant region of mouse kappa or lambda isotype. In some embodiments, the anti- CDKL5 antibody or fragment thereof, comprises a variable light chain region according to any one of SEQ ID NOs.: 82-87, and a light chain constant region of mouse kappa isotype. In some embodiments, the anti-CDKL5 antibody or fragment thereof, comprises a variable light chain region according to any one of SEQ ID NOs.: 82-87, and a light chain constant region of mouse lambda isotype.

[0346] In some embodiments, the anti-CDKL5 antibody or fragment thereof, comprises a heavy chain comprising a variable heavy chain region according to any one of SEQ ID NOs.: 76-81, and a heavy chain constant region of mouse IgG1, mouse IgG2a, mouse IgG2b or mouse IgG3 isotype, and a light chain comprising a variable light chain region according to any one of SEQ ID NOs.: 82-87, and a light chain constant region of mouse kappa or lambda isotype.

[0347] In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain according to SEQ ID NO: 88 and a light chain according to SEQ IDNO: 94. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain according to SEQ ID NO: 89 and a light chain according to SEQ ID NO: 95. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain according to SEQ ID NO: 90 and a light chain according to SEQ ID NO: 96. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain according to SEQ ID NO: 91 and a light chain according to SEQ ID NO: 97. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain according to SEQ ID NO: 92 and a light chain according to SEQ ID NO: 98. In some embodiments, the anti-CDKL5 antibody or a fragment thereof comprises a heavy chain according to SEQ ID NO: 93 and a light chain according to SEQ ID NO: 99. In certain embodiments, In certain embodiments, anti-CDKL5 antibody is a anti-human CDKL5 antibody is an anti-human CDKL5 antibody.

[0348] In some embodiments, the anti-CDKL5 antibody or a fragment thereof is for use in any one of western blot, an enzyme-linked immunosorbent assay (ELISA), radioimmune assay (RIA), immunoprecipitation and immunofluorescence.

[0349] In some embodiments, the anti-CDKL5 antibody or a fragment thereof is linked or coupled to a detectable moiety. In some embodiments, the anti-CDKL5 antibody or a fragment thereof is linked or coupled to any one of an enzyme, a radioisotope and a fluorescent moiety. In some embodiments, the enzyme is a horse radish peroxidase, an alkaline phosphatase, a galactosidase, an acetylcholinesterase, or a catalase. In some embodiments, the fluorescent moiety is a protein fluorophore that is any one of green fluorescent protein (GFP), yellow fluorescent protein (YFP), and red fluorescent protein (RFP) or a modified version thereof. In some embodiments, the fluorescent moiety is a non-protein organic fluorophore that is any one of fluorescein, rhodamine, Oregon green, eosin, Texas red, cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine, BODIPY, aza- BODIPY, squaraine derivative, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, anthraquinones including DRAQ5, DRAQ7, and CyTRAK orange, oxazine derivatives including nile red, nile blue, cresyl violet and oxazine 170, acridine derivatives including proflavin, acridine orange and acridine yellow, and arylmethine derivatives including auramine, crystal violet and malachite green, and tetrapyrrole derivatives including porphin, phthalocyanine and bilirubin. In some embodiments, the radioisotope is any one of 90Y,111In,177Lu,99mTc,123I,125I and131I.

[0350] The detectable moiety can be a luminescent moiety selected from any one of AlexaFluor647, any one of Ruthenium (Ru), fluorescein isothiocyanate (FITC), phycoerythrin (PE) or a conjugate thereof, an Alexa Fluor® dye, a rhodamine dye, a Cy-based dye, a G-dye, or Texas red. The detectable moiety can be a luminescent moiety that is a labelling and conjugating SpyTag / SpyCatcher system known in the art, including but not limited to BioRad SpyCatcher and SpyTag products, Kerafast Biotech SpyTag / SpyCatcher Protein Coupling Reagents or Addgene SpyTag-β-Lactamase-SpyCatcher proteins. In some embodiments, the anti-CDKL5 antibody or a fragment thereof is linked to a SPY peptide (partner 1 peptide) that binds to a corresponding SPY catcher peptide (partner 2 peptide) linked to a detectable label or moiety. In some embodiments, the SPY peptide comprises an amino acid sequence according to SEQ ID NOs: 30-32. In some embodiments, the SPY capture peptide comprises an amino acid sequence according to SEQ ID NOs: 33-35.

[0351] The present disclosure also provides use of SpyTag / SpyCatcher technology to irreversibly conjugate a labeled recombinant SpyCatcher protein to an anti-CDKL5 antibody or a fragment thereof, of the present disclosure.

[0352] In some embodiments, the present disclosure provides a first anti-CDKL5 antibody or fragment thereof that comprises a biotinylable tag according to SEQ ID NO: 14, that can be biotinylated at a single lysine residue in the tag, (antibody biotinylable protein) and a second anti-CDKL5 antibody of fragment thereof, comprising a partner 1 protein tag comprising an amino acid sequence according to any one of SEQ ID NOs: 30-32 (partner 1 protein), that can irreversibly bind a partner 2 protein comprising an amino acid sequence according to any one SEQ ID NOs: 33-35, carrying a detectable label, Ruthenium (Ru). The partner 2 protein can be labeled before conjugation to the anti-CDKL5 antibody partner 1 protein to form a Ru-labelled partner 2 protein-ant-CDKL5 antibody partner 1 protein complex, and can further comprise a His-Tag for purification using a Ni-NTA column. The antibody biotinylable protein labelled anti-CDKL5 antibody can be linked to biotin and then affixed to streptavidin coated plates, followed by incubation with a sample containing CDKL5 protein, wherein CDKL5 protein in the sample can form a tripartite complex with the anti-CDKL5 antibody linked to partner 1 protein and the Ru-labelled partner 2 protein, that be detected from the emitted Ru signal and whereby the level of CDKL5 in the sample is proportional to the intensity of the Ru signal.

[0353] The present disclosure also provides one or more nucleic acid(s) encoding any one of the anti-CDKL5 antibodies or fragments thereof, disclosed herein. In some embodiments, the nucleic acid encodes a heavy chain variable region and comprises a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence according to SEQ ID NO: 100. In some embodiments, the nucleic acid encodes a heavy chain variable region and comprises a nucleotide sequence that is at least 95% identical to a nucleotide sequence according to SEQ ID NO: 100. In some embodiments, the nucleic acid encodes a heavy chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 100. In some embodiments, the nucleic acid encodes a heavy chain variable region and comprises a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence according to SEQ ID NO: 101. In some embodiments, the nucleic acid encodes a heavy chain variable region and comprises a nucleotide sequence that is at least 95% identical to a nucleotide sequence according to SEQ ID NO: 101. In some embodiments, the nucleic acid encodes a heavy chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 101. In some embodiments, the nucleic acid encodes a heavy chain variable region and comprises a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence according to SEQ ID NO: 102. In some embodiments, the nucleic acid encodes a heavy chain variable region and comprises a nucleotide sequence that is at least 95% identical to a nucleotide sequence according to SEQ ID NO: 102. In some embodiments, the nucleic acid encodes a heavy chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 102. In some embodiments, the nucleic acid encodes a heavy chain variable region and comprises a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence according to SEQ ID NO: 103. In some embodiments, the nucleic acid encodes a heavy chain variable region and comprises a nucleotide sequence that is at least 95% identical to a nucleotide sequence according to SEQ ID NO: 103. In some embodiments, the nucleic acid encodes a heavy chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 103. In some embodiments, the nucleic acidencodes a heavy chain variable region and comprises a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence according to SEQ ID NO: 104. In some embodiments, the nucleic acid encodes a heavy chain variable region and comprises a nucleotide sequence that is at least 95% identical to a nucleotide sequence according to SEQ ID NO: 104. In some embodiments, the nucleic acid encodes a heavy chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 104. In some embodiments, the nucleic acid encodes a heavy chain variable region and comprises a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence according to SEQ ID NO: 105. In some embodiments, the nucleic acid encodes a heavy chain variable region and comprises a nucleotide sequence that is at least 95% identical to a nucleotide sequence according to SEQ ID NO: 105. In some embodiments, the nucleic acid encodes a heavy chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 105.

[0354] In some embodiments, the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence according to SEQ ID NO: 106. In some embodiments, the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence that is at least 95% identical to a nucleotide sequence according to SEQ ID NO: 106. In some embodiments, the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 106. In some embodiments, the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence according to SEQ ID NO: 107. In some embodiments, the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence that is at least 95% identical to a nucleotide sequence according to SEQ ID NO: 107. In some embodiments, the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 107. In some embodiments, the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence according to SEQ ID NO: 108. In some embodiments, the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence that is at least 95% identical to a nucleotide sequence according to SEQ ID NO: 108. In some embodiments, the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 108. In some embodiments, the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence according to SEQ ID NO: 109. In some embodiments, the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence that is at least 95% identical to a nucleotide sequence according to SEQ ID NO: 109. In some embodiments, the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 109. In some embodiments, the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence according to SEQ ID NO: 110. In some embodiments, the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence that is at least 95% identical to a nucleotide sequence according to SEQ ID NO: 110. In some embodiments, the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 110. In some embodiments, the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence according to SEQ ID NO: 111. In some embodiments, the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence that is at least 95% identical to a nucleotide sequence according to SEQ ID NO: 111. In some embodiments, the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence according to SEQ ID NO: 111.

[0355] In some embodiments, the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 100, encoding a heavy chain variable region, and a nucleotide sequence according to SEQ ID NO: 106, encoding a light chain variable region. In some embodiments, the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 101, encoding a heavy chain variable region, and a nucleotide sequence according to SEQ ID NO:107, encoding a light chain variable region. In some embodiments, the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 102, encoding a heavy chain variable region, and a nucleotide sequence according to SEQ ID NO: 108, encoding a light chain variable region. In some embodiments, the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 103, encoding a heavy chain variable region, and a nucleotide sequence according to SEQ ID NO: 109, encoding a light chain variable region. In some embodiments, the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 104, encoding a heavy chain variable region, and a nucleotide sequence according to SEQ ID NO: 110, encoding a light chain variable region. In some embodiments, the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 105, encoding a heavy chain variable region, and a nucleotide sequence according to SEQ ID NO: 111, encoding a light chain variable region.

[0356] The present disclosure also provides an expression vector comprising one or more of the nucleic acid(s) disclosed herein. In some embodiments, the expression vector is any one of a plasmid, a viral vector and a cosmid. EXAMPLES

[0357] The disclosure now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to limit the scope of the disclosure in any way.

[0358] CDKL5 is a serine / threonine protein kinase comprising a N-terminal kinase domain, a C-terminal regulatory domain, a NLS1: N-terminal nuclear localization signal, and a NLS2: C-terminal nuclear localization signal (FIG. 1B-1C). The regulatory domain comprises several intrinsically disordered protein (IDP) domains that make up almost two- thirds of the protein (FIGs.1A and 1B), which makes expressing a full-length CDKL5 protein in a mammalian cell very difficult. The studies disclosed herein describe specific tagging strategies for intracellular expression (FIG. 2A-2B), secretion (FIG. 2C-2E) and exosome trafficking (FIG.2F) for expressing and purifying a full-length human CDKL5 protein (FIG. 3).EXAMPLE 1: Optimization of peptide tag linkage and transfection conditions for intracellular expression of rhCDKL5 proteins in a host cell

[0359] The study described herein shows the optimization of specific tag peptide linkage and standardization of the conditions, for optimum expression of a rhCDKL5 protein in a host cell. Expi293 cells were transfected with plasmid DNA encoding either a rhCDKL5 protein comprising (i) an MBP tag linked by a TEV cleavage site at the N-terminus, and a GFP tag linked by a TEV cleavage site at the C-terminus (MBP-TEV-CDKL5-TEV-GFP, FIG.2A), or (ii) a GFP tag linked by a TEV cleavage site at the N-terminus, and an MBP tag linked by a TEV cleavage site at the C-terminus (GFP-TEV-CDKL5-TEV-MBP, FIG.2B). Mock plasmid transfected cells were used as negative control.

[0360] FIGs.4A-4C show increased solubility and expression of the rhCDKL5 protein comprising an intracellular expression tag as described in the present disclosure. The expression level of the rhCDKL5 protein expression in the cells at 48 hours post-transfection was determined by detecting GFP signal using fluorescence microscopy imaging. The results described herein showed that the expression of the GFP-TEV-CDKL5-TEV-MBP (FIG. 4A, right panel) was higher that the expression of the MBP-TEV-CDKL5-TEV-GFP protein (FIG. 4A, middle panel). The level of rhCDKL5 protein in the lysates obtained from the mock plasmid and MBP-TEV-CDKL5-TEV-GFP protein expressing cells was determined by Western-SDS-PAGE and Western blotting with anti-CDKL5 and anti-MBP antibodies (FIG. 4B). The results described herein showed that the expressed rhCDKL5 protein in lysates was degraded, indicating proteolytic cleavage of the protein. Presence of BafA1 (proteasomal degradation pathway inhibitor) in the cell culture post-transfection with the plasmid encoding the rhCDKL5 (MBP-TEV-CDKL5-TEV-GFP) protein did not inh...

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of producing a recombinant human cyclin-dependent kinase-like 5 (rhCDKL5) protein in solution, the rhCDKL5 protein comprising: a CDKL5 sequence comprising an N-terminal catalytic domain and a C-terminal regulatory domain, and at least one intracellular expression tag, wherein the C-terminal regulatory domain comprises a nuclear localization signal 1 domain and a nuclear localization signal 2 domain, the method comprising the steps of: (a) transfecting a host cell culture with a nucleic acid encoding the rhCDKL5 protein; (b) expressing the rhCDKL5 protein in the host cell culture; (c) generating a whole cell lysate by incubating the host cell culture with a lysis buffer; (d) separating soluble fraction comprising the rhCDKL5 protein and insoluble fraction from the whole cell lysate; and (e) isolating the rhCDKL5 protein from the soluble fraction.

2. The method of claim 1, wherein the CDKL5 sequence of the rhCDKL5 protein comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:

2.

3. The method of any one of claims 1-2, wherein the at least one intracellular expression tag is linked to the N-terminus and / or the C-terminus of the rhCDKL5 protein, and wherein the at least one intracellular expression tag is selected from: (a) a green fluorescent protein (GFP) tag; (b) a maltose binding protein (MBP) tag; and (c) a combination thereof.

4. The method of any one of claims 1-3, wherein the rhCDKL5 protein comprises a GFP tag or an MBP tag linked to the N-terminus of the rhCDKL5 protein.

5. The method of any one of claims 1-4, wherein the rhCDKL5 protein comprises a GFP tag or an MBP tag linked to the C-terminus of the rhCDKL5 protein.

6. The method of any one of claims 1-5, wherein the rhCDKL5 protein comprises a GFP tag linked to the N-terminus and an MBP tag linked to the C-terminus of the rhCDKL5 protein, or an MBP tag linked to the N-terminus and a GFP tag linked to the C-terminus of the rhCDKL5 protein.

7. The method of any one of claims 1-6, wherein the at least one intracellular expression tag is linked to the rhCDKL5 protein by a Tobacco etch virus (TEV) protease cleavage site linker.

8. The method of claim 7, wherein an MBP tag is linked to the N-terminus of the rhCDKL5 protein by a TEV protease cleavage site linker and a GFP tag is linked to the C-terminus of the rhCDKL5 protein by a TEV protease cleavage site linker.

9. The method of claim 8, wherein the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO:

4.

10. The method of claim 7, wherein a GFP tag is linked to the N-terminus of the rhCDKL5 protein by a TEV protease cleavage site linker and an MBP tag is linked to the C- terminus of the rhCDKL5 protein by a TEV protease cleavage site linker.

11. The method of claim 10, wherein rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO:

5.

12. The method of any one of claims 1-11, wherein expressing the rhCDKL5 protein in the host cell culture comprises maintaining the host cell culture under conditions adequate to express the rhCDKL5 protein for about 48 hours to about 72 hours.

13. The method of claim 12, wherein maintaining the host cell culture under conditions adequate to express the rhCDKL5 protein comprises maintaining the host cell culture at about 32oC to about 37oC.

14. The method of any one of claims 1-13, wherein expressing the rhCDKL5 protein in the host cell culture comprises maintaining the host cell culture under conditions adequate to express the rhCDKL5 protein in a cell culture medium comprising at least one expression enhancer.

15. The method of claim 14, wherein the at least one expression enhancer comprises an aromatic carboxylic acid, an aliphatic carboxylic acid, a hydroxamic valproic acid, ahydroxamic derivative of valproic acid, an acetamide, sodium butyrate, or a derivative of any one of the foregoing.

16. The method of any one of claims 1-15, wherein the lysis buffer is any one of Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), octylphenoxypolyethoxyethanol (Nonidet P-40), and ammonium-chloride-potassium (ACK) lysis buffer.

17. The method of any one of claims 1-15, wherein the lysis buffer comprises a chelating agent.

18. The method of claim 17, wherein the chelating agent is any one of ethylenediamine- tetraacetic acid disodium salt (EDTA), ethylene glycol-O, O’-bis(2-aminoethyl)-N, N, N’, N’-tetraacetic acid (EGTA), N-(2-Hydroxyethyl)ethylenediamine-N, N’, N’-triacetic acid trisodium salt (HEDTA), nitrilotriacetic acid (NTA), and Potassium Citrate.

19. The method of any one of claims 1-18, wherein isolating the rhCDKL5 protein from the soluble fraction comprises using an affinity-based chromatography resin.

20. The method of claim 19, wherein the affinity-based chromatography resin is a dextrin crosslinked to agarose affinity resin.

21. The method of any one of claims 1-20, wherein the rhCDKL5 protein further comprises a biotinylable peptide tag comprising an amino acid sequence according to SEQ ID NO: 14 at the N-terminus or the C-terminus of the rhCDKL5 protein.

22. The method of claim 21, wherein the biotinylable peptide tag is located between an intracellular expression tag and the C-terminus of the rhCDKL5 protein.

23. The method of claim 22, wherein the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO:

18.

24. The method of any one of claims 21-23, further comprising linking the biotinylable peptide tag of the rhCDKL5 protein to biotin.

25. The method of claim 24, wherein the cells in the host cell culture express an E. coli biotin ligase, BirA.

26. The method of any one of claims 1-20, wherein the rhCDKL5 protein further comprises a partner 1 peptide tag comprising an amino acid sequence according to any one of SEQ ID NOs: 30-32 or a fragment thereof that can bind irreversibly to a partner 2 proteincomprising an amino acid sequence according to any one of SEQ ID NOs: 33-35 or a fragment thereof, and is labelled with a detectable moiety.

27. The method of claim 26, wherein the partner 1 peptide tag or a fragment thereof is linked to the N-terminus of a rhCDKL5 protein comprising an amino acid sequence according to SEQ ID NO:

5.

28. The method of claim 27, wherein the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO:

22.

29. The method of claim 26, wherein the partner 1 peptide tag or a fragment thereof is located between the GFP-TEV linker and the N-terminus of the CDKL5 sequence in the rhCDKL5 protein comprising an amino acid sequence according to SEQ ID NO:

5.

30. The method of claim 29, wherein the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO:

23.

31. The method of claim 26, wherein the partner 1 peptide tag or a fragment thereof is linked to the C-terminus of a rhCDKL5 protein comprising an amino acid sequence according to SEQ ID NO:

5.

32. The method of claim 29, wherein the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO:

24.

33. The method of claim 26, wherein the partner 1 peptide tag or a fragment thereof is located between the C-terminus of the CDKL5 sequence and the MBP-TEV linker in the rhCDKL5 protein comprising an amino acid sequence according to SEQ ID NO:

5.

34. The method of claim 33, wherein the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO:

25.

35. The method of any one of claims 26-34, further comprising a step of contacting the rhCDKL5 protein with a partner 2 protein or a fragment thereof labelled with Ruthenium (Ru).

36. A method of producing a recombinant human cyclin-dependent kinase-like 5 (rhCDKL5) protein in solution, the rhCDKL5 protein comprising: a CDKL5 sequence comprising an N-terminal catalytic domain and a C-terminal regulatory domain, and at least one secretion signal tag; wherein the C-terminal regulatory domain comprises anuclear localization signal 1 domain and a nuclear localization signal 2 domain, the method comprising the steps of: (a) transfecting a host cell culture with a nucleic acid encoding the rhCDKL5 protein; (b) expressing the rhCDKL5 protein in the host cell culture; and (c) separating a supernatant from the host cell culture, wherein the supernatant comprises the expressed rhCDKL5 protein.

37. The method of claim 36, wherein the CDKL5 sequence of the rhCDKL5 protein comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:

2.

38. The method of claims 36 or 37, wherein the at least one secretion signal tag is linked to the N-terminus and / or the C-terminus of the rhCDKL5 protein, and wherein the secretion signal tag is selected from: (a) a kappa light chain signal sequence tag (SEQ ID NO: 6); (b) a heavy chain signal sequence tag (SEQ ID NO: 7); and (c) a hemagglutinin signal sequence tag (SEQ ID NO: 8).

39. The method of any one of claims 36-38, wherein the rhCDKL5 protein further comprises: (a) an affinity purification tag linked to the N-terminus of the rhCDKL5 protein when the rhCDKL5 protein has the at least one secretion signal tag linked to the C-terminus of the rhCDKL5 protein; or (b) an affinity purification tag linked to the C-terminus of the rhCDKL5 protein when the rhCDKL5 protein has the at least one secretion signal tag linked to the N-terminus of the rhCDKL5 protein.

40. The method of claim 39, wherein the affinity purification tag is selected from: (a) a polyhistidine tag; (b) a polyarginine tag; (c) a glutathione-S-transferase tag; (d) a DYKDDDDK (SEQ ID NO: 29) peptide tag; (e) a streptavidin-binding peptide tag;(f) a peptide tag comprising an amino acid sequence according to any one of SEQ ID NOs: 36 or 37; (g) a calmodulin binding peptide tag; (h) a maltose-binding tag; and (i) a cellulose-binding tag.

41. The method of claim 40, wherein the polyhistidine tag is a 6xHIS tag (SEQ ID NO: 21).

42. The method of claim 40 or 41, further comprising purifying the rhCDKL5 protein from the supernatant using any one of: (a) a nickel-nitrilotriacetic acid (Ni-NTA) chromatography resin; (b) a cross linked dextran chromatography resin; (c) an immobilized glutathione resin; (d) an anti-DYKDDDDK (SEQ ID NO: 29) peptide tag antibody resin; (e) a streptavidin resin; (f) a biotin resin; (g) an immobilized chitin resin; (h) a cellulose resin; (i) a maltose resin; and (j) a calmodulin resin.

43. The method of any one of claims 36-42, wherein the rhCDKL5 protein comprises: (a) a kappa light chain signal sequence tag linked to the N-terminus of the rhCDKL5 protein and a 6xHIS tag linked to the C-terminus of the rhCDKL5 protein; (b) a heavy chain signal sequence tag linked to the N-terminus of the rhCDKL5 protein and a 6xHIS tag linked to the C-terminus of the rhCDKL5 protein; or (c) a hemagglutinin signal sequence tag linked to the N-terminus of the rhCDKL5 protein and a 6xHIS tag linked to the C-terminus of the rhCDKL5 protein.

44. The method of claim 43, wherein the rhCDKL5 protein of (a), (b), and (c) comprises an amino acid sequence according to SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, respectively.

45. The method of any one of claims 36-44, wherein expressing the rhCDKL5 protein in the host cell culture comprises maintaining the host cell culture under conditions adequate to express the rhCDKL5 protein for a duration of up to 7 days.

46. The method of claim 45, wherein maintaining the host cell culture under conditions adequate to express the rhCDKL5 protein comprises maintaining the culture at about 32oC to about 37oC.

47. The method of claim 45 or 46, wherein the step of separating the supernatant comprising the expressed rhCDKL5 protein from the host cell culture is repeated or is conducted continuously throughout the duration of expressing the rhCDKL5 protein in the host cell culture.

48. A method of producing a recombinant human cyclin-dependent kinase-like 5 (rhCDKL5) protein in solution, the rhCDKL5 protein comprising: a CDKL5 sequence comprising an N-terminal catalytic domain and a C-terminal regulatory domain, and an exosome trafficking tag; wherein the C-terminal regulatory domain comprises a nuclear localization signal 1 domain and a nuclear localization signal 2 domain, the method comprising the steps of: (a) transfecting a host cell culture with a nucleic acid encoding the rhCDKL5 protein; (b) expressing the rhCDKL5 protein in the host cell culture; and (c) separating a supernatant from the host cell culture, wherein the supernatant comprises exosomes comprising the expressed rhCDKL5 protein.

49. The method of claim 48, wherein the CDKL5 sequence of the rhCDKL5 protein comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:

2.

50. The method of claim 48 or 49, wherein the exosome trafficking tag is an exosome- targeting viral peptide, or a fragment thereof.

51. The method of claim 50, wherein the exosome trafficking tag is linked to the N-terminus or the C-terminus of the rhCDKL5 protein and is selected from: (a) an HIV GAG protein or a fragment thereof; (b) an HIV NEF peptide; (c) an HTLV-1 transactivator protein (Tax); (d) an HCV core protein; and (e) an HCV glycoprotein E2.

52. The method of claim 51, wherein the exosome trafficking tag is an HIV GAG protein or a fragment thereof.

53. The method of claim 52, wherein the HIV GAG protein or a fragment thereof is linked to the N-terminus of the rhCDKL5 protein.

54. The method of any one of claims 48-53, wherein the rhCDKL5 protein further comprises an affinity purification tag.

55. The method of claim 54, wherein the affinity purification tag is linked to the exosome trafficking tag.

56. The method of claim 55, wherein the affinity purification tag is selected from: (a) a polyhistidine tag; (b) a polyarginine tag; (c) a glutathione-S-transferase tag; (d) a DYKDDDDK (SEQ ID NO: 29) peptide tag; (e) a streptavidin-binding peptide tag; (f) a peptide tag comprising an amino acid sequence according to any one of SEQ ID NOs: 36 or 37; (g) a calmodulin binding peptide tag; (h) a maltose-binding tag; and (i) a cellulose-binding tag.

57. The method of claim 56, wherein the polyhistidine tag is a 6xHIS tag (SEQ ID NO: 21).

58. The method of claim 56 or 57, further comprising purifying the rhCDKL5 protein from the supernatant using any one of: (a) a nickel-nitrilotriacetic acid (Ni-NTA) chromatography resin;(b) a cross linked dextran chromatography resin; (c) an immobilized glutathione resin; (d) an anti-DYKDDDDK (SEQ ID NO: 29) peptide tag antibody resin; (e) a streptavidin resin; (f) a biotin resin; (g) an immobilized chitin resin; (h) a cellulose resin; (i) a maltose resin; and (j) a calmodulin resin.

59. The method of any one of claims 48-58, wherein the affinity purification tag and / or the exosome trafficking tag is linked to the N-terminus or C-terminus of the rhCDKL5 protein by a Tobacco etch virus (TEV) protease cleavage site linker.

60. The method of claim 59, wherein the rhCDKL5 protein comprises an HIV GAG protein or a fragment thereof and a 6xHIS tag linked by a linker peptide, and wherein the 6xHIS tag is linked to the N-terminus of the rhCDKL5 protein by a TEV protease cleavage site linker.

61. The method of claim 60, wherein the rhCDKL5 protein comprises an amino acid sequence according to SEQ ID NO:

13.

62. The method of any one of claims 48-61, wherein expressing the rhCDKL5 protein in the host cell culture comprises maintaining the host cell culture under conditions adequate to express the rhCDKL5 protein for a duration of up to 7 days.

63. The method of claim 62, wherein maintaining the host cell culture under conditions adequate to express the rhCDKL5 protein comprises maintaining the culture at about 32oC to about 37oC.

64. The method of claim 62 or 63, wherein the step of separating the supernatant comprising the exosomes comprising the expressed rhCDKL5 protein from the host cell culture is repeated or conducted continuously throughout the duration of expressing the rhCDKL5 protein in the host cell culture.

65. Use of the rhCDKL5 protein or a fragment thereof produced by the method of any one of claims 1-64, in an analytical method for detecting a CDKL5-specific antibody in a sample.

66. The use of claim 65, wherein the CDKL5-specific antibody is an anti-drug-antibody (ADA) raised in a subject administered a therapeutic CDKL5 gene or protein.

67. The use of claim 65 or 66, wherein the analytical method is any one of a colorimetric method, enzyme immunoassay (EIA), radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLIA), and a counting immunoassay (CIA).

68. The use of any one of claims 65-67, wherein the analytical method comprises the steps of: (a) immobilizing the rhCDKL5 protein or a fragment thereof to a solid surface; (b) contacting the immobilized rhCDKL5 protein with the sample for an amount of time sufficient for binding of the immobilized rhCDKL5 protein to the CDKL5-specific antibody, if present in the sample, to form a bipartite complex of the rhCDKL5 protein and the CDKL5-specific antibody; (c) contacting the bipartite complex with a detection reagent that binds specifically to the CDKL5-specific antibody and not the rhCDKL5 protein, for an amount of time to form a tripartite complex of the detection reagent, the rhCDKL5 protein, and the CDKL5-specific antibody, wherein the detection reagent comprises a detectable moiety that generates a detectable signal; and (d) detecting the signal generated in step (c), wherein presence of the detectable signal indicates presence of CDKL5-specific antibody in the sample, and absence of the detectable signal indicates absence of CDKL5-specific antibody in the sample.

69. The use of claim 65, wherein the rhCDKL5 protein or fragment thereof is immobilized to the solid surface by an anti-CDKL5 antibody bound to the solid surface or by covalent crosslinking to the solid surface.

70. The use of claim 69, wherein the solid surface is any one of polyethylene, polyacrylamide, polystyrene, agarose, glass, and silicone rubber.

71. The use of claim 70, wherein the analytical method further comprises the steps of: (i) contacting the immobilized rhCDKL5 protein of step (a) of claim 68 with a coating / blocking buffer before proceeding with step (b) of claim 68; (ii) contacting the bipartite complex of step (b) of claim 68 with a wash buffer before proceeding with step (c) of claim 68; (iii) contacting the tripartite complex of step (c) of claim 68 with a wash buffer before proceeding with step (d) of claim 68; (iv) contacting the tripartite complex of step (c) of claim 68 with a detection solution before proceeding with step (d) of claim 68; or (v) a combination thereof.

72. The use of any one of claims 65-71, wherein the detectable moiety is any one of an enzyme, a fluorophore, and a radioisotope.

73. The use of claim 72, wherein the detectable signal is any one of a colorimetric signal, a fluorescent signal, and radiation.

74. The use of claim 72, wherein the detectable moiety is an enzyme, and step (c) of claim 68 further comprises contacting the tripartite complex with a substrate for generating the detectable signal.

75. The use of claim 74, wherein the enzyme is any one of a horse radish peroxidase, an alkaline phosphatase, a galactosidase, an acetylcholinesterase, and a catalase.

76. The use of claim 72, wherein the fluorophore is any one of fluorescein isothiocyanate (FITC), phycoerythrin (PE) or a conjugate thereof, an Alexa Fluor®dye, a rhodamine dye, a Cyanine-based dye, an Orange G-dye, and sulforhodamine 101 acid chloride dye.

77. The use of claim 72, wherein the radioisotope is any one of90Y,111In,177Lu,99mTc123I, 125I, and131I.

78. The use of any one of claims 65-77, wherein the detection reagent is an antibody or a fragment thereof.

79. The use of any one of claims 65-78, wherein the rhCDKL5 protein or a fragment thereof to be immobilized to the solid surface is a rhCDKL5 protein produced by the method of any one of claims 21-23.

80. The use of claim 79, wherein the rhCDKL5 protein is a biotinylated rhCDKL5 protein.

81. The use of claim 80, wherein the biotinylated rhCDKL5 protein is immobilized by contacting on a solid surface coated with streptavidin.

82. The use of any one of claims 65-71 and 79-81, wherein the detection reagent is a rhCDKL5 protein produced by the method of any one of claims 26-35.

83. The use of claim 82, further comprises a step of contacting the rhCDKL5 protein with a partner 2 protein or a fragment thereof labeled with Ruthenium (Ru), before contacting the bipartite complex with a detection reagent.

84. Use of the rhCDKL5 protein or a fragment thereof produced by the method of any one of claims 1-64, in an analytical method of quantitatively measuring an amount of CDKL5 present in a sample solution.

85. The use of claim 84, wherein the analytical method is any one of a colorimetric method, enzyme immunoassay (EIA), radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLIA), and counting immunoassay (CIA).

86. The use of claim 84 or 85, wherein the analytical method comprises the steps of: (a) contacting the sample solution with a CDKL5 capture reagent and a CDKL5 detection reagent, and detecting a sample signal from the CDKL5 detection reagent in the sample solution; (b) measuring the sample signal; (c) contacting one or more reference solutions, each comprising a known amount of the rhCDKL5 protein, with the CDKL5 capture reagent and the CDKL5 detection reagent, and detecting one or more comparator signals from the CDKL5 detection reagent in each of the one or more solutions; (d) measuring the one or more comparator signals; and(e) comparing the sample signal with the one or more comparator signals to quantitatively determine the concentration of CDKL5 in the sample solution.

87. The use of claim 86, wherein the CDKL5 capture reagent has binding specificity for CDKL5 in the sample solution and for the rhCDKL5 protein or a fragment thereof in the reference solutions.

88. The use of claim 87, wherein the CDKL5 capture reagent is an antibody or a fragment thereof.

89. The use of any one of claims 86-88, wherein the CDKL5 capture reagent is immobilized to a solid surface.

90. The use of claim 89, wherein the CDKL5 capture reagent is immobilized by a protein A to a solid surface.

91. The use of any one of claims 86-90, wherein the CDKL5 detection reagent binds specifically to CDKL5 in the sample solution and the rhCDKL5 protein or a fragment thereof in the reference solutions but does not bind to the CDKL5 capture reagent.

92. The use of claim 91, wherein the CDKL5 detection reagent is an antibody or a fragment thereof.

93. The use of any one of claims 86-90, wherein the CDKL5 detection reagent used in step (a) of claim 86 is different from the CDKL5 detection reagent used in step (c) of claim 86.

94. The use of any one of claims 86-93, wherein detecting a signal from the CDKL5 detection reagent comprises contacting the CDKL5 detection reagent with a secondary detection reagent that specifically binds the CDKL5 detection reagent and not the CDKL5 capture reagent.

95. The use of claim 94, wherein the secondary detection reagent is an antibody or a fragment thereof.

96. The use of claim 95, wherein the secondary detection reagent comprises a reporter moiety.

97. The use of claim 96, wherein the reporter moiety is any one of a horse radish peroxidase, an alkaline phosphatase, a galactosidase, an acetylcholinesterase, and a catalase.

98. The use of any one of claims 86-97, wherein the analytical method further comprises contacting the sample solution and the one or more reference solutions comprising known amounts of the rhCDKL5 protein with a washing solution before measuring the sample signal.

99. The use of any one of claims 65-98, wherein the sample is from a subject suffering from or suspected of having a CDKL5 deficiency disorder, CDKL5 encephalopathy, CDKL5- related epilepsy, CDKL5-related epileptic encephalopathy, early infantile epileptic encephalopathy 2, optionally wherein the subject has been administered or is to be administered a therapeutic CDKL5 gene or protein.

100. The use of any one of claims 65-99, wherein the sample is a tissue homogenate, a cell lysate, or other biological fluid from a subject.

101. A kit for detecting CDKL5-specific antibody in a sample, comprising: (a) a recombinant human CDKL5 (rhCDKL5) protein or a fragment thereof, produced by the method of any one of claims 1-64; and (b) a detection reagent that specifically binds to the CDKL5-specific antibody but not to the rhCDKL5 protein or fragment thereof, wherein the detection reagent comprises a detectable moiety.

102. The kit of claim 101, further comprising: (a) a solid surface; (b) a coating / blocking solution; (c) a washing solution; (d) a detection solution; or (e) a combination thereof.

103. The kit of claim 102, wherein the rhCDKL5 protein or fragment thereof is immobilized to the solid surface.

104. The kit of claim 102 or 103, wherein the rhCDKL5 protein or a fragment thereof to be immobilized to the solid surface is a rhCDKL5 protein of any one of claims 21-23.

105. The kit of claim 104, wherein the rhCDKL5 protein is a biotinylated rhCDKL5 protein.

106. The kit of claim 105, wherein the biotinylated rhCDKL5 protein is immobilized on a solid surface coated with streptavidin.

107. The kit of any one of claims 101-106, wherein the detection reagent is a rhCDKL5 protein of any one of claims 26-35.

108. The kit of claim 107, comprising a partner 2 protein or a fragment thereof labeled with Ruthenium (Ru) for forming a complex with the rhCDKL5 protein, before contacting the bipartite complex with a detection reagent.

109. The kit of claim 102, further comprising an anti-CDKL5 antibody or a crosslinker for immobilizing the rhCDKL5 protein or fragment thereof to the solid surface.

110. The kit of claim 109, wherein the solid surface is coated with protein A agarose.

111. The kit of claim 110, wherein the solid surface is any one of polyethylene, polyacrylamide, polystyrene, agarose, glass, and silicone rubber.

112. The kit of any one of claims 101-111, wherein the detectable moiety is any one of an enzyme, a fluorophore, and a radioisotope.

113. The kit of claim 112, wherein the detectable moiety generates a detectable signal which is any one of a colorimetric signal, a fluorescent signal, and radiation.

114. The kit of claim 112, wherein the detectable moiety is an enzyme, and the kit further comprises a substrate that generates a detectable signal upon reacting with the enzyme.

115. The kit of claim 114, wherein the enzyme is any one of a horse radish peroxidase, an alkaline phosphatase, a galactosidase, an acetylcholinesterase, and a catalase.

116. The kit of claim 112, wherein the fluorophore is any one of fluorescein isothiocyanate (FITC), phycoerythrin (PE) or a conjugate thereof, an Alexa Fluor®dye, a rhodamine dye, a Cyanine-based dye, an Orange G-dye, and sulforhodamine 101 acid chloride dye.

117. The kit of claim 112, wherein the radioisotope is any one of90Y,111In,177Lu,99mTc 123I,125I, and131I.

118. The kit of any one of claims 101-102 and 112-117, wherein the detection reagent is an antibody or a fragment thereof.

119. A kit for quantitatively measuring an amount of CDKL5 present in a sample solution comprising:(a) a CDKL5 capture reagent; (b) a CDKL5 detection reagent; and (c) a standard solution comprising a recombinant human CDKL5 (rhCDKL5) protein produced by the method of any one of claims 1-64 or a fragment thereof, or a lyophilized reagent for preparing said standard solution.

120. The kit of claim 119, wherein the CDKL5 detection reagent binds specifically to CDKL5 in the sample solution and the rhCDKL5 protein or a fragment thereof in the standard solution.

121. The kit of claim 120, wherein the CDKL5 detection reagent is an antibody or a fragment thereof that specifically binds to rhCDKL5 protein.

122. The kit of any one of claims 119-120, further comprising: (a) a solid surface; (b) a coating solution; (c) a washing solution; (d) a detection solution; or (e) a combination thereof.

123. The kit of any one of claims 119-122, wherein the CDKL5 capture reagent binds specifically to CDKL5 in the sample solution and the rhCDKL5 protein or a fragment thereof in the standard solution.

124. The kit of claim 123, wherein the CDKL5 capture reagent is an antibody or a fragment thereof that specifically binds to CDKL5 at a site that is different from the binding site of the CDKL5 detection reagent.

125. The kit of claim 124, wherein the CDKL5 capture reagent is immobilized to the solid surface.

126. The kit of claim 125, wherein the CDKL5 capture reagent is immobilized by protein A to the solid surface.

127. The kit of any one of claims 119-126, wherein the CDKL5 detection reagent binds specifically to CDKL5 in the sample solution and the rhCDKL5 protein or a fragment thereof in the standard solution but does not bind to the CDKL5 capture reagent.

128. The kit of any one of claims 119-127, wherein the CDKL5 detection reagent is different from the CDKL5 capture reagent.

129. The kit of any one of claims 119-128, further comprising a secondary detection reagent that specifically binds the CDKL5 detection reagent and not the CDKL5 capture reagent.

130. The kit of claim 129, wherein the secondary detection reagent is an antibody or a fragment thereof.

131. The kit of any one of claims 129-130, wherein the secondary detection reagent comprises a reporter moiety.

132. The kit of claim 131, wherein the reporter moiety is any one of a horse radish peroxidase, an alkaline phosphatase, a galactosidase, an acetylcholinesterase, and a catalase.

133. A method of producing a full-length, non-denatured, soluble, and tagless recombinant human CDKL5 protein, the method comprising the steps of: a) providing a recombinant human CDKL5 (rhCDKL5) protein linked to one or more intracellular expression tags or exosome trafficking tag linked by a TEV protease cleavage site linker to the N- and C-terminus of the rhCDKL5 protein, produced by any one of the methods of claims 7-35 and 59-61; b) incubating the rhCDKL5 protein with an effective amount of a TEV protease to remove the TEV protease cleavage site linker and generate a reaction mixture comprising a tagless recombinant human CDKL5 protein and impurities comprising cleaved one or more intracellular expression peptide tags or one or more exosome trafficking tags and partially cleaved rhCDKL5 proteins; c) applying the reaction mixture to an immobilized-metal affinity chromatography (IMAC) resin to generate a flowthrough comprising the impurities; and d) applying an elution buffer to the IMAC resin to generate an eluent comprising the tagless recombinant human CDKL5 protein.

134. The method of claim 133, wherein the method further comprises passing the eluent comprising the tagless recombinant human CDKL5 protein through a hydrophobicinteraction chromatography resin to generate a second eluent comprising the tagless recombinant human CDKL5 protein.

135. A method of expressing CDKL5 protein in a subject in need thereof, by administering to the subject a therapeutically effective amount of a human recombinant CDKL5 protein generated by the method of claim 133 or 134.

136. The method of claim 135, wherein the subject is a mammal.

137. The method of claim 136, wherein the mammal is a human, a non-human primate, or a rodent.

138. The method of any one of claims 135-137, further comprising detecting the presence of human CDKL5 in the subject by: i) obtaining a sample from the subject; ii) contacting the sample with one or more proteolytic enzyme(s) for an amount of time sufficient to generate one or more peptide fragments; and iii) detecting the presence of a human specific CDKL5 sequence in the one or more peptide fragments of step ii).

139. The method of claim 138, wherein the one or more proteolytic enzyme(s) is selected from the group consisting of trypsin, chymotrypsin, Lys-C endoproteinase, AspN endoproteinase, GluC endoproteinase, and proteinase K.

140. The method of claim 139, wherein the one or more proteolytic enzyme(s) comprises Lys-C endoproteinase and chymotrypsin.

141. The method of any one of claims 138-140, wherein the presence of a human specific CDKL5 sequence in the one of more peptide fragments is detected by any one of a mass spectrometry (MS), colorimetric method, enzyme immunoassay (EIA), radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLIA), and counting immunoassay (CIA).

142. The method of claim 141, wherein the mass spectrometry is a liquid chromatography- mass spectrometry (LC-MS).

143. The method of claim 142, wherein the LC-MS comprises any one of adsorption chromatography, partition chromatography, ion-exchange chromatography, size- exclusion chromatography, and affinity chromatography.

144. The method of any one of claims 138-143, wherein the human specific CDKL5 sequence comprises an amino acid sequence according to SEQ ID NO:

28.

145. An anti-CDKL5 antibody or fragment thereof that binds to a CDKL5 protein according to any one of SEQ ID Nos: 1-2, 4-5, 9-11, 13, 15-18 and 22-27.

146. The anti-CDKL5 antibody or fragment thereof of claim 145, wherein the antibody or fragment thereof binds to the kinase domain of the CDKL5 protein.

147. The anti-CDKL5 antibody or fragment thereof of claim 145, wherein the antibody or fragment thereof binds to the non-catalytic / non-kinase domain of the CDKL5 protein.

148. The anti-CDKL5 antibody or fragment thereof of any one of claims 145-147, wherein the antibody or fragment thereof comprises any one of: (a) a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 42, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 43 and a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 44; (b) a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 45, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 46 and a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 47; (c) a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 48, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 49 and a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 50; (d) a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 51, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 52 and a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 53; (e) a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 54, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 55 and a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 56; and(f) a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 57, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 58 and a HCDR3 comprising an amino acid sequence according to SEQ ID NO:

59.

149. The anti-CDKL5 antibody or fragment thereof of any one of claims 145-148, wherein the antibody or fragment thereof comprises any one of: (a) a LCDR1 comprising an amino acid sequence according to SEQ ID NO: 60, a LCDR2 comprising an amino acid sequence of LVS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 62; (b) a LCDR1 comprising an amino acid sequence according to SEQ ID NO: 63, a LCDR2 comprising an amino acid sequence of KVS and a LCDR3 according to SEQ ID NO: 65; (c) a LCDR1 comprising an amino acid sequence according to SEQ ID NO: 66, a LCDR2 comprising an amino acid sequence of KVS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 67; (d) a LCDR1 comprising an amino acid sequence according to SEQ ID NO: 68, a LCDR2 comprising an amino acid sequence of LVS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 69; (e) a LCDR1 comprising an amino acid sequence according to SEQ ID NO: 70, a LCDR2 comprising an amino acid sequence of WAS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 72; and (f) a LCDR1 comprising an amino acid sequence according to SEQ ID NO: 73, a LCDR2 comprising an amino acid sequence of QVS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO:

75.

150. The anti-CDKL5 antibody or fragment thereof of any one of claims 145-148, wherein the antibody or fragment thereof comprises any one of: (a) a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 42, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 43, a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 44, LCDR1 comprising an amino acid sequence according to SEQ ID NO: 60, aLCDR2 comprising an amino acid sequence of LVS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 62; (b) a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 45, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 46, a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 47, LCDR1 comprising an amino acid sequence according to SEQ ID NO: 63, a LCDR2 comprising an amino acid sequence of KVS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 65; (c) a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 48, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 49, a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 50, LCDR1 comprising an amino acid sequence according to SEQ ID NO: 66, a LCDR2 comprising an amino acid sequence of KVS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 67; (d) a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 51, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 52, a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 53, LCDR1 comprising an amino acid sequence according to SEQ ID NO: 68, a LCDR2 comprising an amino acid sequence of LVS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 69; (e) a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 54, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 55, a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 56, LCDR1 comprising an amino acid sequence according to SEQ ID NO: 70, a LCDR2 comprising an amino acid sequence of WAS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO: 72; and (f) a HCDR1 comprising an amino acid sequence according to SEQ ID NO: 57, a HCDR2 comprising an amino acid sequence according to SEQ ID NO: 58, a HCDR3 comprising an amino acid sequence according to SEQ ID NO: 59, LCDR1 comprising an amino acid sequence according to SEQ ID NO: 73, aLCDR2 comprising an amino acid sequence of QVS and a LCDR3 comprising an amino acid sequence according to SEQ ID NO:

75.

151. The anti-CDKL5 antibody or fragment thereof of any one of claims 145-148, wherein the antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence of any one of SEQ ID Nos: 76-81.

152. The anti- CDKL5 antibody or fragment thereof of any one of claims 145-151, wherein the antibody or fragment thereof comprises any one of: (a) a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 76; (b) a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 77; (c) a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 78; (d) a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 79; (e) a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 80; and (f) a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO:

81.

153. The anti-CDKL5 antibody or fragment thereof of any one of claims 145-148, wherein the antibody or fragment thereof comprises a light chain variable region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence of any one of SEQ ID Nos: 82-87.

154. The anti-CDKL5 antibody or fragment thereof of any one of claims 145-148, wherein the antibody or fragment thereof comprises any one of: (a) a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 82; (b) a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 83;(c) a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 84; (d) a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 85; (e) a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 86; and (f) a light chain variable region comprising an amino acid sequence according to SEQ ID NO:

87.

155. The anti- CDKL5 antibody or fragment thereof of any one of claims 145-154, wherein the antibody or fragment thereof comprises any one of: (a) a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 76, and a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 82; (b) a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 77, and a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 83; (c) a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 78, and a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 84; (d) a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 79, and a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 85; (e) a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 80, and a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 86; and (f) a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 81, and a light chain variable region comprising an amino acid sequence according to SEQ ID NO:

87.

156. The anti-CDKL5 antibody or fragment thereof of any one of claims 145-148, wherein the antibody or fragment thereof comprises a heavy chain comprising an amino acidsequence that is at least 95% identical to an amino acid sequence of any one of SEQ ID Nos: 88-93.

157. The anti- CDKL5 antibody or fragment thereof of any one of claims 145-148, wherein the antibody or fragment thereof comprises any one of: (a) a heavy chain comprising an amino acid sequence according to SEQ ID NO: 88; (b) a heavy chain comprising an amino acid sequence according to SEQ ID NO: 89; (c) a heavy chain comprising an amino acid sequence according to SEQ ID NO: 90; (d) a heavy chain comprising an amino acid sequence according to SEQ ID NO: 91; (e) a heavy chain comprising an amino acid sequence according to SEQ ID NO: 92; and (f) a heavy chain comprising an amino acid sequence according to SEQ ID NO:

93.

158. The anti-CDKL5 antibody or fragment thereof of any one of claims 145-148, wherein the antibody or fragment thereof comprises a light chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence of any one of SEQ ID Nos: 94-99.

159. The anti-CDKL5 antibody or fragment thereof of any one of claims 145-148, wherein the antibody or fragment thereof comprises any one of: (a) a light chain comprising an amino acid sequence according to SEQ ID NO: 94; (b) a light chain comprising an amino acid sequence according to SEQ ID NO: 95; (c) a light chain comprising an amino acid sequence according to SEQ ID NO: 96; (d) a light chain comprising an amino acid sequence according to SEQ ID NO: 97;(e) a light chain comprising an amino acid sequence according to SEQ ID NO: 98; and (f) a light chain comprising an amino acid sequence according to SEQ ID NO:

99.

160. The anti-CDKL5 antibody or fragment thereof of any one of claims 145-148, wherein the antibody or fragment thereof comprises any one of: (a) a heavy chain comprising an amino acid sequence according to SEQ ID NO: 88, and a light chain comprising an amino acid sequence according to SEQ ID NO: 94; (b) a heavy chain comprising an amino acid sequence according to SEQ ID NO: 89, and a light chain comprising an amino acid sequence according to SEQ ID NO: 95; (c) a heavy chain comprising an amino acid sequence according to SEQ ID NO: 90, and a light chain comprising an amino acid sequence according to SEQ ID NO: 96; (d) a heavy chain comprising an amino acid sequence according to SEQ ID NO: 91, and a light chain comprising an amino acid sequence according to SEQ ID NO: 97; (e) a heavy chain comprising an amino acid sequence according to SEQ ID NO: 92, and a light chain comprising an amino acid sequence according to SEQ ID NO: 98; and (f) a heavy chain comprising an amino acid sequence according to SEQ ID NO: 93, and a light chain comprising an amino acid sequence according to SEQ ID NO:

99.

161. One or more nucleic acid(s) encoding the anti- CDKL5 antibody or fragment thereof of any one of claims 145-160.

162. The one or more nucleic acid(s) of claim 161, wherein the one or more nucleic acid(s) encodes a heavy chain variable region and comprises a nucleotide sequence that is at least 95% identical to a nucleotide sequence of any one of SEQ ID Nos: 100-105.

163. The one or more nucleic acid(s) of claim 161 or 162, wherein the one or more nucleic acid(s) encodes a heavy chain variable region and comprises a nucleotide sequence according to any one of: (a) SEQ ID NO: 100; (b) SEQ ID NO: 101; (c) SEQ ID NO: 102; (d) SEQ ID NO: 103; (e) SEQ ID NO: 104; and (f) SEQ ID NO:

105.

164. The one or more nucleic acid(s) of any one of claims 161-163, wherein the nucleic acid encodes a light chain variable region and comprises a nucleotide sequence that is at least 95% identical to a nucleotide sequence of any one of SEQ ID Nos: 106-111.

165. The one or more nucleic acid(s) of any one of claims 161-164, wherein the one or more nucleic acid(s) encodes a light chain variable region and comprises a nucleotide sequence according to any one of: (a) SEQ ID NO: 106; (b) SEQ ID NO: 107; (c) SEQ ID NO: 108; (d) SEQ ID NO: 109; (e) SEQ ID NO: 110; and (f) SEQ ID NO:

111.

166. The one or more nucleic acid(s) of any one of claims 161-165, wherein the one or more nucleic acid(s) comprises any one of: (a) a nucleotide sequence according to SEQ ID NO: 100 encoding a heavy chain variable region, and a nucleotide sequence according to SEQ ID NO: 106 encoding a light chain variable region; (b) a nucleotide sequence according to SEQ ID NO: 101 encoding a heavy chain variable region, and a nucleotide sequence according to SEQ ID NO: 107 encoding a light chain variable region;(c) a nucleotide sequence according to SEQ ID NO: 102 encoding a heavy chain variable region, and a nucleotide sequence according to SEQ ID NO: 108 encoding a light chain variable region; (d) a nucleotide sequence according to SEQ ID NO: 103 encoding a heavy chain variable region, and a nucleotide sequence according to SEQ ID NO: 109 encoding a light chain variable region; (e) a nucleotide sequence according to SEQ ID NO: 104 encoding a heavy chain variable region, and a nucleotide sequence according to SEQ ID NO: 110 encoding a light chain variable region; and (f) a nucleotide sequence according to SEQ ID NO: 105 encoding a heavy chain variable region, and a nucleotide sequence according to SEQ ID NO: 111 encoding a light chain variable region.

167. An expression vector comprising the one or more nucleic acid(s) of any one of claims 161-166.

168. The expression vector of claim 167, wherein the expression vector is any one of a plasmid, a viral vector, and a cosmid.

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