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Definitions

• CDKL5 proteins and gene therapy compositions which can be used to treat CDKL5 -mediated neurological disorders such as a CDKL5 deficiency or an atypical Rett syndrome caused by a CDKL5 mutation or deficiency.
• Other aspects of the invention pertain to methods of producing such recombinant CDKL5 proteins and gene therapy compositions, as well as pharmaceutical compositions, methods of treatment, and uses of such recombinant proteins and gene therapy compositions.
• the composition further comprises one or more of an SV40 intron, a polyadenylation signal or a stabilizing element.
• the polynucleotide encoding the leader signal polypeptide has at least 90% sequence identity to SEQ ID NO: 169.
• Another aspect of the present invention relates to a pharmaceutical formulation comprising a composition as described herein and a pharmaceutically acceptable carrier.
• Another aspect of the present invention relates to a method of treating a
• Another aspect of the present invention relates to a pharmaceutical formulation comprising a CDKL5 polypeptide or fusion protein as described herein and a pharmaceutically acceptable carrier.
• the fusion protein further comprises one or more affinity-tags, one or more protease cleavage sites, or combinations thereof.
• the affinity-tag comprises MYC, HA, V5, NE, StrepII, Twin-Strep-tag®, glutathione S-transferase (GST), maltose-binding protein (MBP), calmodulin-binding peptide (CBP), FLAG®, 3xFLAG®, polyhistidine (His), HPC4, or combinations thereof.
• the protease cleavage site is sensitive to one or more of thrombin, furin, factor Xa, metalloproteases, enterokinases, cathepsin, HRV3C, TEV, or combinations thereof.
• Figure 7 shows a Western blot of a CDKF5 fusion protein that was coexpressed in the cytoplasm of HEK293F with several potential substrates.
• Figure 12B shows a Sypro Ruby Red stained gel demonstrating HRV3C protease cleavage of the CDKL5 fusion protein of Figure 11 A.
• CDKL5-HRV3C-FLAG-His-HPC4 protein CDKL5-HRV3C-FLAG-His-HPC4 protein.
• CDKL5 fusion proteins CDKL5 fusion proteins
• CDKL5 polypeptides e.g wild-type CDKL5 polypeptides, CDKL5 variants with one or more N-linked glycosylation sites removed and/or shorter CDKL5 variants.
• CDKL5 phosphorylates methyl-CpG binding protein 2 (MeCP2), and independent loss-of-function mutations in MeCP2 lead to the Rett syndrome phenotype.
• Other substrates of CDKL5 include Netrin G1 ligand (NGL-1), Shootinl (SHTN1), Mindbomb 1 (MIB1), DNA (cytosine-5)-methyltransferase 1 (DNMT1), Amphiphysin 1 (AMPH1), end binding protein EB2, microtubule associated protein IS (MAP1S) and histone deacetylase 4 (HDAC4).
• CDKL5b (Chen el al.). In general, there is a high level of sequence conservation in CDKL5 genes across human, rat, and mouse species except for the last 100-150 amino acids near the C- terminus. Western blot data show that both variants are present during rat development yet adults appear to predominately express a single variant. Furthermore, CDKL5 is present in identifiable quantities in brain, liver, and lung.
• CDKL5 has also been shown to phosphorylate the protein DNA methyltransferase 1 (DNMT1) (Kameshita I, et al. “Cyclin-dependent kinase-like 5 binds and phosphorylates DNA methyltransferase 1.” Biochem Biophys Res Commun 377:1162-1167).
• DNMT1 DNA methyltransferase 1
• This phosphorylation leads to activation of DNMT1 which is a maintenance-type methylation protein that preferentially methylates hemimethylated DNA. This process is useful for maintenance of DNA methylation patterns during DNA replication, so that newly synthesized daughter DNA strands are able to maintain the methylation pattern of the parent strand it replaced.
• DNMT1 DNA methyltransferase 1
• the CDKL5 polypeptide may contain deletions, substitutions and/or insertions relative to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more deletions, substitutions and/or insertions to the amino acid sequence described by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.
• the CDKL5 polypeptide has at least 98%, at least 98.5%, at least 99% or at least 99.5% sequence identity to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25.
• the CDKL5 polypeptide comprises one or more protease cleavage sites.
• the protease cleavage site is located on one or more of the N-terminus or the C-terminus of the CDKL5 polypeptide.
• Exemplary protease cleavage sites include, but are not limited to, cleavage sites sensitive to thrombin, furin, factor Xa, metalloproteases, enterokinases, cathepsin, HRV3C, TEV, and combinations thereof.
• TAT translocate across the plasma membrane
• Recent work has explored the possibility that a special type of endocytosis is involved with TAT uptake, and a few cell lines have been identified that appear resistant to TAT penetration.
• the specific cargo to be delivered by TAT may also play a role in the efficacy of delivery.
• Previous research data have suggested that a TAT fusion protein has better cellular uptake when it is prepared in denaturing conditions, because correctly folded protein cargo likely requires much more energy (delta-G) to cross the plasma membrane due to structural constraints.
• the CDKL5 variants described herein are operably linked to a CPP such as TAT, modified TAT (TATK), Transportan, Antennapedia or P97.
• TAT can refer to the original TAT peptide having 11 amino acids (designated TAT11) or can refer to a TAT peptide having an additional 16 N-terminal amino acids (designated as TAT28) that are derived from the polylinker of the plasmid used for cloning.
• TAT can refer to a modified version of TATI 1 (designated TATKI 1) or a modified version of TAT28 (designated TATK28).
• the CPP can have an N-terminal glycine added.
• TATK28 and TAT28 would otherwise have an N-terminal aspartate residue, which has a low stability.
• Adding an N-terminal glycine to the sequence can increase protein stability via the N-end rule.
• any of the fusion proteins that have a leader signal polypeptide can have a glycine added at the C-terminal end of the leader signal polypeptide, such that upon cleavage of the leader signal polypeptide, the new N-terminus of the fusion protein will begin with glycine.
• the CPP is operatively coupled to the N-terminus of the CDKL5 polypeptide. In one or more embodiments, the CPP is operatively coupled to the C-terminus of the CDKL5 polypeptide. [00116] In one or more embodiments, the CPP comprises one or more affinity-tags. In one or more embodiments, the affinity-tag is located on one or more of the N-terminus or the C-terminus of the CPP. Examples of affinity-tags that can be added to the CPP include, but are not limited to, epitope tags ( e.g .
• CDKL5 variants can be used in fusion proteins, such as proteins that also contain a CPP.
• Other polypeptides can also be incorporated into such fusion proteins, such as leader signal polypeptides to enhance protein secretion or affinity-tags for detecting and/or purifying the fusion proteins, as well as linker polypeptides that can be used to link functional polypeptides.
• the fusion protein comprises a CDKL5 polypeptide having an N-terminal CPP, optionally with a leader signal polypeptide before the N-terminal CPP. In one or more embodiments, the fusion protein comprises a CDKL5 polypeptide having a C-terminal CPP, optionally with a leader signal polypeptide before the CDKL5 polypeptide. In one or more embodiments, the fusion protein comprises a leader signal peptide and a CDKL5 polypeptide without a CPP. [00121] Examples of affinity-tags that can be added to the fusion proteins include, but are not limited to, epitope tags ( e.g .
• Some embodiments of the fusion protein may also include a protease cleavage site.
• the protease cleavage site is located on the N-terminus of affinity- tag.
• the protease cleavage site is located on the C-terminus of affinity- tag.
• Exemplary protease cleavage sites include, but are not limited to, cleavage sites sensitive to thrombin, furin, factor Xa, metalloproteases, enterokinases, cathepsin, HRV3C, TEV and combination thereof.
• the recombinant protein (e.g. CDKL5 variant or fusion protein) can be expressed in and secreted from host cells using appropriate vectors.
• mammalian cells e.g., CHO, HeLa or HEK cells
• insect cells e.g. Sf9 or BTI-Tn-5Bl-4
• bacterial cells e.g., E. coli or P. haloplanktis TAC 125 cells
• Exemplary plasmids are described in the examples below and shown in Figures 2A-2BK.
• Figure 10 shows relative CDKL5 expression and yield in bacterial, mammalian and insect cell expression system.
• the CDKL5 variant or the fusion protein has a Twin-Strep- tag®.
• the CDKL5 variant or the fusion protein with the affinity-tag is purified on a purification resin.
• the purification resin is a strep-tactin resin.
• the CDKL5variant or the fusion protein may also include one or more protease cleavage sites.
• the protease cleavage site is located on the N-terminus of the CDKL5 variant or the fusion protein.
• the protease cleavage site is located on the C-terminus of the CDKL5 variant or the fusion protein.
• the protease cleavage site is located on N-terminus and C- terminus of the CDKL5 variant or the fusion protein.
• the cleavage is performed when the CDKL5 variant or the fusion protein is bound to the purification resin.
• the cleavage is performed when the CDKL5 variant or the fusion protein with the Twin-Strep-tag® is bound to the strep-tactin resin.
• kits containing the gene therapy composition e.g. comprising CDKL5 polynucleotides
• protein replacement therapy composition e.g.
• Figure 2BE shows an exemplary plasmid for expressing the fusion protein of SEQ ID NO: 99 in CHO cells.
• This fusion protein comprises the modified BiP leader signal polypeptide, TATK28 and the 1-5, 7NQ CDKL5 IO7 glycosylation variant.
• Figure 2BK shows an exemplary plasmid for expressing the fusion protein of SEQ ID NO: 105 in CHO cells.
• This fusion protein comprises the modified BiP leader signal polypeptide, TATK28 and the 1-9NQ CDKL5 IO7 glycosylation variant.
• Example 4 Methotrexate Amplification of CDKL5 Fusion Proteins in CHO Cells
• Methotrexate amplification was used to amplify expression of CDKL5 fusion proteins in CHO-DG44 cells.
• TATK28-CDKL5_107-FH no signal sequence
• Igk-TATk28- CDKL5_107-FH Igk-TATk28- CDKL5_107-FH
• mBiP-TATK28-CDKL5_107-FH were cloned into the pOptiVec vector providing the DHFR gene for methotrexate resistance.
• plasmids were transfected into DG44 cells (deficient in dhfr ) and selected by growth in medium deficient in hypoxanthine and thymidine.
• Methotrexate-resistant subcultures were obtained by culturing the cells sequentially in 0.1, 0.25, 0.5, and 1 m M Methotrexate (MTX), allowing cells to recover to 70% viability between steps.
• Cell pellets were lysed in 50 mM Tris-HCl with 75 mM NaCl, 1% Triton X- 100, and 1.5X protease inhibitor cocktail (EDTA-free), pH 7.4. 40 pg of total protein were resolved on LDS-PAGE, transferred to nitrocellulose membranes, probed with a rabbit antipolyhistidine antibody (Thermo), and detected with a fluorescent secondary antibody.
• the TATK28-eGFP-CDKL5 construct only provided a secreted quantity of CDKL5 fusion protein of about 0.1 pg/L, while the hiB ⁇ R-TATk28- CDKL5 construct achieved a secreted quantity of CDKL5 fusion protein of about 15 pg/L (a 150-fold increase).
• the CDKL5 fusion proteins represented 0.1% (1 mg/g) of total protein.
• Example 6 Co-expression of CDKL5 Fusion Proteins and Potential Substrates
• pCHO 1.0 harboring both TATK28-CDKL5-FH (no signal sequence) and one of several putative CDKL5 substrates (HOMER 1, HDAC4, ARHGEF2, MAPRE2, AMPH1, or SHANK1), or no protein partner, were transiently transfected into HEK293F cells. After five days in culture, cells were harvested and lysed in 50 mM sodium phosphate, 150 mM sodium chloride, 0.5% Triton-X100, IX Complete Protease Inhibitor Complex, EDTA-free, pH 7 for 30 minutes at 4 °C.
• Blot shown in Figure 4A demonstrates that the fusion protein comprising the wild-type CDKF5 IO7 isoform is highly glycosylated when expressed in CHO-DG44 cells prior to treatment with PNGase F, whereas substituting 7 of the Asn residues of the N-linked glycosylation sites with Gin (1-7NQ) produces a fusion protein with little to no glycosylation when expressed in the CHO-DG44 cells.
• fusion proteins comprising the CDKF5 glycosylation variants 1-4, 6-7NQ; 1-5, 7NQ; 1-6NQ; 2NQ; 2-7NQ; 1, 3-7NQ; 1-2, 4-7NQ and 1-3, 5-7NQ were expressed in HEK293F cells, and untreated or treated with PNGase F and are shown in Figure 4B.
• These fusion proteins comprising the other glycosylation variants had varying degrees of glycosylation and were all less glycosylated than the fusion protein comprising the wild-type CDKF5 IO7 isoform, thus showing that the various N-linked glycosylation sites can be glycosylated in isolation. Fusion proteins comprising the wild-type CDKF5iis isoform were also found to be glycosylated.
• Sf9 cells were co-transfected with linearized baculovims (BV) DNA and transfer plasmids: 1) GST-P-TATK28-eGFP-P-FH; 2) GST-P-eGFP-P-FH; 3) GST-P-TAT28-CDKL5_107-P-FH; 4) GST-P-TATK28-CDKL5_107-P-FH; 5) GST-P-p97p-CDKL5_107-P-FH; 6) GST-P-Antp- CDKL5_107-P-FH; 7) GST-P-TAT11-CDKL5_107-P-FH and GST-P-Transp-CDKL5_107-P- FH (coding sequences being Sf9 codon-optimized), lpg protein run out on duplicate 4-12%, 10-well NuPage gels. Gels run at 175V for 90 minutes. Protein transferred to nitrocellulose using the iBLOT at 20v for
• CDKL5 fusion proteins from insect cells were also purified to isolate the CDKL5 proteins from the cell lysate.
• GST-P-TATK28-CDKL5_107-P-FH proteins were expressed in High Five (BTI-Tn-5Bl-4) cells maintained as suspension cultures in Sf900II media.
• Infected cell pellets were lysed with 50 mM NaPOzj, 500 mM NaCl, 10% Glycerol, pH 6) supplemented with IX HALT Protease Inhibitor cocktail without EDTA (Thermo, 78437), 1 mM tris 2-carboxyethyl-phosphine (TCEP) and 5 mM EDTA at a ratio of 10 ml Lysis Buffer per 100 million cells.
• Triton X-100 was added to 0.5%. The lysate was clarified by centrifugation at 31,000 x g for 20 minutes.
• the soluble material was adjusted to 350 mM NaCl and applied to HiTrap SP Fast Flow resin (GE Healthcare, 17-5157-01). Bound protein was eluted with a 10 column volume (CV) NaCl gradient, 350-2000 mM. The CDKL5 protein peak, 525-1225 mM NaCl, was buffer-exchanged in to Buffer B (50 mM NaPOzj, 500 mM NaCl, 10% Glycerol, IX HALT Protease inhibitor cocktail without EDTA, 1 mM TCEP, pH 8). Protein was applied to IMAC Sepharose 6 FF resin (GE Healthcare, 17-0921-09) that had been charged with Nickel Sulfate and pre-equilibrated with Buffer B.
• the resin was washed with Buffer B + 60 mM imidazole.
• the resin with incubated with 40 U of HRV3C protease (Millipore, 71493) at 4 °C up to overnight to remove the GST, FLAG and polyhistidine (His) affinity tags .
• Aliquots of the cleaved material examined at 3hours and overnight.
• the resin was washed with 50 mM NaP04, 500 mM NaCl, 10% Glycerol, 1 mM TCEP + IX HALT PI- EDTA + 0.5% Triton X-100 + 500 mM imidazole to elute the CDKL5.
• the eluted protein lacks the affinity tags and migrates more quickly though SDS-PAGE.
• Figures 10A and 10B show a Sypro Ruby Red total protein stained gel analysis.
• Figure 11A shows the expression of GST-P-TATK28-CDKL5_107-P-FH in insect cells compared to uninfected control cells and the recovery of tagged protein on the IMAC resin.
• Figure 11B shows the tagged CDKF5 protein prior to and post- cleavage with the eluted protein from the IMAC resin.
• Figure 12A shows a Sypro Ruby Red stained gel of a CDKF5 fusion protein in cell lysate and the purified fusion protein.
• Figure 12B shows a Sypro Ruby Red stained gel demonstrating HRV3C protease cleavage of the CDKF5 fusion protein of Figure 11A
• GST-P-TATK28-CDKF5_107-P-FH expressed in HighFive cells via infection with baculovims was released from cells by lysis in 50 mM Na-phosphate, 500 mM NaCl, 10% glycerol, 1 mM TCEP, 1 mM EDTA, 1 x HAFT protease inhibitor cocktail, pH 6.0, using nitrogen cavitation for 15 minutes at room temperature. Following cell disruption, Triton X- 100 was added to 0.5%, and incubated for 30 minutes at 4 °C. The lysate was separated into soluble and insoluble fractions by centrifugation at 15,000 x g for 15 minutes at room temperature. The soluble fraction was then further modified with the following conditions by dilution to the same final volume:
• FIG. 13 shows that the CDKL5 fusion protein is soluble at high salt concentrations (e.g at least 500 mM NaCl) and NaCl levels lower than 500 mM result in insoluble CDKL5 protein.
• the CDKL5 protein can be briefly exposed to NaCl concentrations as low at 350 mM, but some loss in incurred. For this reason, most purification steps described herein are carried out in high salt levels, but such high salt levels may be incompatible with in vivo administration.
• the insoluble pellet was washed with the lysis buffer. The washed insoluble pellet was then resuspended in 2 ml of the lysis buffer and sonicated. The soluble fraction after sonication was used for protein analysis. BCA assay was used to measure the protein concentration. NuPAGE was used to analyze the protein expression in insect cells. In Figure 18, start and load shows total cellular protein and soluble fraction respectively.
• Cultured embryonic primary cortical neurons were treated with 10 pg/ml recombinant TATK28-CDKL5 for 6 hours. Non-treated cultured embryonic primary cortical neurons were used as a negative control. Each sample, either treated or non-treated, were fixed in 4% PFA, permeabilized in 0.1% saponin, and stained using anti-MAP2, anti-CDKL5, and/or anti-phosphorylated (S222) EB2 antibodies. The cells were counterstained with DAPI and mounted on glass microscope slides under Prolong Diamond anti-fade mounting medium. The samples were imaged using a Leica SP8 point scanning laser confocal microscope with a 63x oil-immersion objective.
• Figure 20D-20F shows results of the uptake experiment, where the cells were treated with TATK28-CDKL5.
• Figure 20D shows image of rat DIV14 embryonic primary cortical neurons stained with anti-DAPI and anti- MAP2 under the fluorescence microscope.
• Figure 20E is an enlarged section of Figure 20D.
• Figure 20F shows Figure 20E but only for anti-CDKL5 fluorescence.
• Similar experiments were also performed in rat DIV7 embryonic primary cortical neurons to compare the results with rat DIV14 embryonic primary cortical neurons.
• Figure 21A-21F shows the uptake of TATK28-CDKL5 in rat DIV7 embryonic primary cortical neurons.
• Figure 22A-22F shows the uptake of TATK28-CDKL5 in rat DIV14 embryonic primary cortical neurons.
• Figure 22A-22C represent images of negative controls.
• Figure 22A shows image of embryonic primary cortical neurons stained with anti-DAPI, anti- MAP2 and anti-CDKL5 protein under the fluorescence microscope
• Figure 22B is an enlarged section of Figure 22A.
• Figure 22C shows Figure 22B but only for DAPI and anti-CDKL5 protein fluorescence.
• Figure 22D-22F shows results of the uptake experiment, where the cells were treated with the TATK28-CDKL5 protein.
• Figure 22D shows image of rat DIV14 embryonic primary cortical neurons stained with anti-DAPI, anti-MAP2 and anti-CDKL5 protein under the fluorescence microscope.
• Figure 22E is an enlarged section of Figure 22D.
• Figure 22F shows Figure 22E but only for DAPI and anti-CDKL5 protein fluorescence.
• the cultured embryonic primary cortical neurons were treated with 10 pg/ml recombinant TATK28-CDKL5 for 15 min, 30 min, 2 hr, 6 hr, or 24 hours.
• treated coverslips were fixed in 4% PFA, permeabilized in 0.1% saponin, and stained using anti-MAP2, anti-CDKL5, and/or anti- phosphorylated (S222) EB2 antibodies.
• the cells were counterstained with DAPI and mounted on glass microscope slides under Prolong Diamond anti-fade mounting medium. The samples were imaged using a Leica SP8 point scanning laser confocal microscope with a 63x oil- immersion objective.
• FIG. 23A-23J An analysis of Figure 23A-23J indicates TATK28-CDKL5 protein accumulation in cortical neurons that increases gradually increase in signal intensity over a period of at least 6 hours.
• Analysis of phospho (S222) EB2 signal was performed using ImageJ software and graphed with GraphPad Prism software.
• Figure 24 observe an increase in intensity of phospho (S222) EB2 signal following uptake, an indication that the TATK28-CDKL5 is active inside the cell.
• CDKL5 protein is reported to co-localize with PSD95 in neurons.
• the DIV14 neurons were treated with 15 pg/rnl of TATK28-CDKL5 for 2 hours. The neurons were then stained with anti-PSD95 and anti-CDKL5.
• Figure 25 A and Figure 25B shows co-localization of CDKL5 with PSD95 and Synapsinl respectively.
• SEQ ID NOS: 106-121 provide exemplary sequences for CDKL5 AAV vectors.
• SEQ ID NO: 106 provides an exemplary sequence for a plasmid for expressing the full-length human CDKL5 IO7 isoform using the CBh promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28. The DNA sequence is codon-optimized for expression in mice.
• SEQ ID NO: 113 provides an exemplary sequence for a plasmid for expressing a fusion protein comprising a modified BiP leader signal polypeptide, TATK28, NLS and eGFP using the CBh promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28.
• the DNA sequence is codon-optimized for expression in mice.
• SEQ ID NO: 114 provides an exemplary sequence for a plasmid for expressing the full-length human CDKL5 IO7 isoform using the hSynl promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28.
• the DNA sequence is codon-optimized for expression in mice.
• SEQ ID NO: 115 provides an exemplary sequence for a plasmid for expressing a kinase-dead version of the full-length human CDKL5 IO7 isoform using the hSynl promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28.
• the DNA sequence is codon-optimized for expression in mice.
• SEQ ID NO: 120 provides an exemplary sequence for a plasmid for expressing a fusion protein comprising a modified BiP leader signal polypeptide, TATK28 and eGFP using the hSynl promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28.
• the DNA sequence is codon-optimized for expression in mice.
• SEQ ID NO: 122 An exemplary DNA sequence codon-optimized for expression of a fusion protein in a human is provided in SEQ ID NO: 122.
• the fusion protein encoded by SEQ ID NO: 122 comprises a modified BiP leader signal polypeptide, TATK28 and the full-length human CDKL5 IO7 isoform.
• Exemplary DNA sequences for TATKI I, TATK28, Antennapedia, Transportan and P97 that are codon-optimized for human expression (but without the initiator methionine codon or the stop codon) are provided in SEQ ID NOS: 150-154, respectively.
• Exemplary DNA sequences for TATKK28 that are codon-optimized for human expression (but without the initiator methionine codon or the stop codon) using different codon optimization tools are provided in SEQ ID NOS: 170-173
• An exemplary DNA sequence for mBIP that is codon-optimized for human expression (including the initiator methionine codon but without the stop codon) is provided in SEQ ID NO: 155.
• An exemplary DNA sequence for mvBIP that is codon-optimized for human expression (including the initiator methionine codon but without the stop codon) is provided in SEQ ID NO: 169.
• FIG. 27-29 shows anti-NeuN antibody, anti-CDKL5 RNA riboprobe and anti- CDKL5 protein antibody stained images of striatum, thalamus and hippocampal formation regions of brains, respectively.
• FIG. 29A and 29B represents image of immunostained brain section from the control group
• Figure 29C and 29D represents image of immunostained brain section from the treatment group
• Figure 29A and 29C represents image of brain section stained with DAPI, anti-NeuN and anti-CDKF5 protein.

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