HK1226745A1 - Tatk-cdkl5 fusion proteins, compositions, formulations, and use thereof - Google Patents

Abstract

Disclosed herein are compositions and formulations containing a TATk-CDKL5 fusion protein. Also disclosed are methods of producing a TATk-CDKL5 fusion protein from vectors containing a TATk-CDKL5 cDNA and methods of transducing cells with the vectors containing a TATk-CDKL5 cDNA and the TATk-CDKL5 fusion protein

Description

TATk-CDKL5fusion protein, composition, preparation and application thereof

Cross Reference to Related Applications

The present application claims the benefit OF U.S. provisional patent application serial No. 61/946,280, filed on 28.2.2014, having the title "TATk-CDKL 5FUSION PROTEINS, COMPOSITIONS, FORMULATIONS, AND METHODS OF making AND USING them (TATk-CDKL5FUSION PROTEINS, COMPOSITIONS, FORMULATIONS, AND the material METHODS OF MAKING AND METHODS OF USING)", the disclosure OF which is incorporated herein in its entirety.

One or more sequence Listing

This application contains a sequence listing submitted in electronic form as an ascii. txt file entitled 02158765.txt, created 2 months and 27 days 2015, and having a size of 84,059 bytes. The contents of this sequence listing are incorporated herein in their entirety.

Background

Cyclin-dependent kinase-like 5(CDKL5) mutations/defects (also known as atypical Rett syndrome) are a debilitating postnatal nervous system disorder that occurs in 1 worldwide every 17000 to 38000 women born. Men are also affected at a lower incidence. The condition is not limited to human or ethnic origin. Symptoms of CDKL5 mutation/deficiency range from mild to severe and are present as early seizures, cognitive disability, hypotonia, and autonomic, sleep, and gastrointestinal disorders. Symptoms of the disease are caused by a defect in the functional CDKL5 protein.

Mutations in the X-chromosome associated CDKL5 gene or defects in the CDKL5 protein in individuals have been implicated in the development of atypical or congenital Rett syndrome. See Beltani et al, J.biol. chem.). 2006,281: 32048-. The CDKL5 gene is located on the X chromosome and encodes proteins essential for normal brain development and function. The CDKL5 protein is a multifunctional protein with pleiotropic effects in neuronal cells. For example, CDKL5 may act as a kinase and phosphorylate MeCP 2. Girls affected by a CDKL5 mutation or defect typically have the following characteristics: normal prenatal history; irritability and drowsiness during perinatal period; early onset epilepsy, which occurs 5 months ago; rett-like features including head growth retardation, stereotypy, poor to lost voluntary hand use, and sleep disturbances, and severe mental retardation with poor eye communication, and almost speech loss. See bachibie (Bahi-Buisson) and benfubi (Bienvenu), 2012 molecular synthesis (mol. synthol.) 2: 137-.

Current treatments for CDKL5 mutations/defects are focused primarily on treating symptoms. However, currently there is no treatment that improves neurological outcome in subjects with CDKL5 mutations or defects. Therefore, there is a need to develop therapies for treating CDKL5 mutations and defects.

SUMMARY

Described herein are fusion proteins having a CDKL5 polypeptide sequence and a TATk polypeptide sequence, wherein the CDKL5 polypeptide sequence has about 50% to 100% sequence identity to SEQ ID No. 2 or SEQ ID No. 16, wherein the TATk polypeptide sequence has about 90% to about 100% sequence identity to SEQ ID No. 4, wherein the TATk polypeptide is operably linked to the CDKL5 polypeptide. In some aspects, the fusion protein can comprise an Igk-chain leader sequence polypeptide, wherein the Igk-chain leader sequence is operably linked to the CDKL5 polypeptide. In further aspects, the fusion protein can contain a reporter protein polypeptide, wherein the reporter protein polypeptide is operably linked to the CDKL5 polypeptide. In other aspects, the fusion protein can contain a protein tag polypeptide, wherein the protein tag polypeptide is operably linked to the CDKL5 polypeptide. In some aspects, the fusion proteins can increase neurite growth, neurite elongation, number of neurite branches, or density of neurite branches in the brain of a subject, as compared to a control. In other aspects, the fusion proteins can reduce neuronal apoptosis in the brain of a subject compared to a control. In some aspects, the fusion protein can have a polypeptide sequence according to SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12 or SEQ ID NO 14.

Also provided herein are pharmaceutical formulations containing a therapeutically effective amount of a fusion protein having a CDKL5 polypeptide sequence and a TATk polypeptide sequence, wherein the CDKL5 polypeptide sequence has about 50% to 100% sequence identity to SEQ ID No. 2 or SEQ ID No. 16, wherein the TATk polypeptide sequence has about 90% to about 100% sequence identity to SEQ ID No. 4, wherein the TATk polypeptide is operably linked to the CDKL5 polypeptide, and a pharmaceutically acceptable carrier. In some aspects, the fusion protein included in the pharmaceutical formulations can contain an Igk-chain leader sequence polypeptide, wherein the Igk-chain leader sequence is operably linked to the CDKL5 polypeptide. In some aspects, the fusion protein included in the pharmaceutical formulations can contain a reporter protein polypeptide, wherein the reporter protein polypeptide is operably coupled to the CDKL5 polypeptide. In further aspects, the fusion protein comprised in the pharmaceutical formulations may have a polypeptide sequence according to SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12 or SEQ ID NO 14. In further aspects, the therapeutically effective amount of the fusion protein can treat one or more symptoms of a CDKL5 deficiency, Rett syndrome, or a Rett syndrome variant in a subject, as compared to a control. In additional aspects, the therapeutically effective amount of the fusion protein can increase neurite growth, neurite elongation, neurite branch number or neurite branch density in the brain of a subject, as compared to a control. In other aspects, the therapeutically effective amount of the fusion protein can reduce neuronal apoptosis in the brain of the subject compared to a control. In a further aspect, the therapeutically effective amount of the fusion protein can improve motor function in a subject compared to a control. In a further aspect, the therapeutically effective amount of the fusion protein can improve cognitive function in a subject compared to a control.

Provided herein are methods of administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical formulation containing an amount of a fusion protein and a pharmaceutically acceptable carrier, wherein the fusion protein contains a CDKL5 polypeptide sequence and a TATk polypeptide sequence, wherein the CDKL5 polypeptide sequence has about 50% to 100% sequence identity to SEQ ID No. 2 or SEQ ID No. 16, wherein the TATk polypeptide sequence has about 90% to about 100% sequence identity to SEQ ID No. 4, wherein the TATk polypeptide is operably linked to the CDKL5 polypeptide. In some aspects, the subject in need thereof has or is suspected of having a CDKL5 deficiency, Rett syndrome, or a Rett syndrome variant. In other aspects of the method of administering a therapeutically effective amount of the pharmaceutical formulation, the therapeutically effective amount of the fusion protein can treat one or more symptoms of a CDKL5 deficiency, Rett syndrome, or a Rett syndrome variant in a subject, as compared to a control.

Brief Description of Drawings

Figure 1 shows one embodiment of a method of producing a CDKL5fusion protein, wherein the CDKL5fusion protein is produced by cultured cells and secreted into the surrounding medium.

Figure 2 shows an embodiment of a method of producing a CDKL5fusion protein, wherein the CDKL5fusion protein is not secreted into the surrounding cell culture medium.

Figure 3 illustrates one embodiment of a method of delivering CDKL5fusion protein via autologous cells.

Fig. 4A and 4B show results of western blot analysis of TATk-CDKL5 protein expression from transfected HEK293T cells. TATk-CDKL5fusion proteins were labeled with GFP proteins to allow western blot analysis using anti-GFP antibodies. Figure 4A shows TATk-GFP-CDKL 5fusion protein expression in cell extracts from transfected HEK293T cells. Figure 4B shows TATk-GFP-CDKL 5fusion protein purification from TATk-GFP-CDKL 5-transfected HEK293T cells by 20X concentrated cell culture medium.

Fig. 5A and 5B show results from kinase activity assays (fig. 5A), demonstrating that the TAT-GFP-CDKL 5fusion protein retains the autophosphorylation activity of CDKL 5.

Figure 6 shows the effect of incubation time on the transduction efficiency of one embodiment of the TATk-GFP-CDKL 5fusion protein in HEK293T cells.

Fig. 7A and 7B show the localization of CDKL5 in TATk-GFP-CDKL5 treated HEK293T cells (fig. 7B). Fig. 7A and 7B demonstrate the efficiency of transduction of HEK293T cells with TATk-GFP-CDKL 5fusion protein compared to the control (fig. 7A) (left panel). Immunodetection was performed using anti-GFP antibody and cells were counterstained with DAPI. White arrows indicate transduced HEK293T cells.

FIG. 8 is an image showing a series of 12 images (1-12) from a confocal microscope showing TATk-GFP-CDKL5 transduction into SH-SY5Y cells treated with purified TATk-GFP-CDKL5 protein for 30 minutes. The Z stack size was 0.4 μm. FIG. 8 shows the transduction efficiency of SH-SY5Y cells with TATk-GFP-CDKL 5fusion protein.

Fig. 9A and 9B show the effect of transduced CDKL5 on cell proliferation in neuroblastoma cells (SH-SY 5Y). Decreased proliferation was observed in TATk-GFP-CDKL5 treated cells (fig. 9B) compared to TATk-GFP (control) treated cells (fig. 9A). White arrows indicate mitotic nuclei.

FIG. 10 shows a graph demonstrating mitotic index of SH-SY5Y cells treated with TATk-GFP or TATk-GFP-CDKL 5fusion proteins. The y-axis shows mitotic cells/total cells and is expressed as a percentage. Data are shown as mean ± s.e. P <0.001 (t-test).

FIGS. 11A-11B are images showing representative phase-contrast images of TATk-GFP treated (control) SH-SY5Y cells (FIG. 11A) and TATk-GFP-CDKL5 treated SH-SY5Y cells (FIG. 11B). Greater neurite outgrowth was observed in TATk-GFP-CDKL5 treated SH-SY5Y cells compared to control cells.

FIG. 12 shows a graph demonstrating quantification of neurite outgrowth of SH-SY5Y cells treated with TATk-GFP fusion protein (control) or TATk-GFP-CDKL 5. Data are shown as mean ± s.e. P <0.05 (t-test). The y-axis shows neurite (neurotic) length/cell in microns.

Figures 13A-13B show images showing dendritic morphology and number of neonatal hippocampal granulocytes as shown by immunohistochemistry of biscortin (DCX) in wild type (figure 13A) and CDKL5 knock-out (KO) mice (figure 13B). Scale bar 50 μm. Abbreviations: GR, granular layer; h, a door.

Figures 14A-14B show dual fluorescence images of differentiated Neuronal Precursor Cells (NPC) demonstrating reduced production and maturation of new neurons (red cells) in neuronal cultures derived from CDKL5 knockout mice (-/-) (figure 14A) compared to wild type (+/+) (figure 14B) neuronal cultures. Cells with neuronal phenotype were immunopositive for β -tubulin III (red) and cells with astrocytic phenotype were immunopositive for GFAP (green). Nuclei were stained with Hoechst dye (blue). Scale bar 25 μm.

Fig. 15A-15C show representative images of neuronal precursor cultures (fig. 15B and 15C) from CDKL5 knockout mice transduced with TATk-GFP (fig. 15B) or TATk-GFP-CDKL5 (fig. 15C), and neuronal precursor cultures (fig. 15A) from wild type mice. Scale bar 20 μm.

Figure 16 shows a graph demonstrating quantification of neural maturation as measured by total neurite length of differentiated neurons (neurons positive for β -tubulin III) in neuronal precursor cultures from wild type and CDKL5KO mice treated with TATk-GFP or TATk-GFP-CDKL 5. Values represent mean ± SE. P <0.01 compared to wild type case; compared to untreated KO samples, # p <0.01(ANOVA followed by bonflorni (Bonferroni) test).

Figures 17A-17F show images showing immunodetection of CDKL5 in the brain of mice (day 7 after birth) treated systemically (single injection) with concentrated medium (vehicle) (figures 17A and 17D), TATk-GFP (figures 17B and 17E), and TATk-GFP-CDKL5 (figures 17C and 17F). Fig. 17D to 17F show enlarged views of the broken-line boxes in fig. 17A to 17C, respectively. Localization of TATk-GFP-CDKL5 and TATk-GFP in brain was assessed by immunohistochemistry using anti-GFP antibody (red). The images are taken at the level of the sensory-motor cortex. Scale bar 60 μm (lower magnification) and 20 μm (higher magnification).

Figures 18A-18D show images of immunodetected cerebellar sections displaying CDKL5 in the brain of mice (day 7 after birth) treated systemically with medium (vehicle) (figure 18A and figure 18B) and TATk-GFP-CDKL5 (figure 18C and figure 18D) as in figures 17A-17F. Localization of TATk-GFP-CDKL5 in brain was assessed by immunohistochemistry using anti-GFP antibody (fig. 18A-18B). Slides were fixed with DAPI to stain nuclei (fig. 18B, 18C). Abbreviations: EGL, outer granular layer; IGL, inner granular layer; ML, molecular layer; PL, Purkinje (Purkinje) layer. Scale bar 60 μm.

Figure 19 demonstrates placement of a cannula administered intracerebroventricularly to mice for TATk-GFP-CDKL 5fusion protein.

Fig. 20 shows a sketch depicting the implant and fusion protein injection schedules for the studies illustrated in fig. 21-33.

Figures 21A-21C show images of DCX immunostained hippocampal dentate gyral sections demonstrating a reduction in neurite length and number of neonatal granulosa cells in CDKL5 knockout mice compared to wild type mice (figure 21B and figure 21A, respectively). It was observed that intraventricular administration of TATk-GFP-CDKL 5fusion protein continuously for five days increased neurite length and number of neonatal granulosa cells in CDKL5 knockout mice (fig. 21C) to levels similar to wild-type (fig. 21A). Scale bar 70 μm.

Fig. 22A-22C show enlarged views of the images at the level of the layer of granules indented back in fig. 21. Scale bar 25 μm.

FIGS. 23A-23B show examples of reconstituted dendritic trees of novacells of: wild type (+/Y) (fig. 23A), CDKL5 knockout mice (-/Y) (fig. 23B) and CDKL5 knockout male mice treated with TATk-GFP-CDKL 5fusion protein via intraventricular injections given once daily for 5 consecutive days (-/Y + TATk-GFP-CDKL5) (fig. 23C).

Figures 24A-24B show graphs of quantification of the average total dendritic length (figure 24A) and average number of dendritic segments (figure 24B) of dentate gyroplastic cells (DCX positive cells) displaying: wild type male mice (+/Y), CDKL5 knockout mice (-/Y), and CDKL5 knockout male mice treated with TATk-GFP-CDKL 5fusion protein via intraventricular injection given once daily for 5 consecutive days (-/Y + TATk-GFP-CDKL 5). Values represent mean ± SE. P < 0.01; p <0.001 compared to +/Y; compared to the-/Y samples, # p <0.05(ANOVA followed by Ponferenb test).

Figures 25A-25B show graphs showing quantification of the average length (figure 25A) and average number (figure 25B) of branches of different stages of dentate gyroparticulate cells displaying: wild type male mice (+/Y), CDKL5 knockout mice (-/Y), and CDKL5 knockout male mice treated with TATk-GFP-CDKL 5fusion protein via intraventricular injection given once daily for 5 consecutive days (-/Y + TATk-GFP-CDKL 5). Values represent mean ± SE. P < 0.05; p <0.01 compared to +/Y; compared to the-/Y samples, # p <0.05(ANOVA followed by Ponferenb test).

Figure 26 shows a graph demonstrating quantification of apoptotic cells (caspase-3 positive cells) in: wild type male mice (+/Y), CDKL5 knockout male mice (-/Y), and CDKL5 knockout male mice treated with TATk-GFP-CDKL 5fusion protein via intraventricular injection given once daily for 5 consecutive days (-/Y + TATk-GFP-CDKL 5). Values represent mean ± SE. P <0.05 compared to +/Y; compared to the-/Y samples, # p <0.05(ANOVA followed by Ponferenb test).

Figure 27 shows a graph of quantification of DCX positive cell number in DG displaying: wild type male mice (+/Y), CDKL5 knockout male mice (-/Y), and CDKL5 knockout male mice treated with TATk-GFP-CDKL 5fusion protein via intraventricular injection given once daily for 5 consecutive days (-/Y + TATk-GFP-CDKL 5). Data are expressed as number of cells/mm 2, p <0.05 compared to +/Y; compared to the-/Y samples, # p <0.05(ANOVA followed by Ponferenb test).

Fig. 28A-28C show representative images of brain sections processed for Synaptophysin (SYN) immunofluorescence displaying a layer of Dentate Gyrus (DG) molecules from: wild type male mice (+/Y) (fig. 28A), CDKL5 knockout male mice (-/Y) (fig. 28B), and CDKL5 knockout male mice treated with TATk-GFP-CDKL 5fusion protein via intraventricular injection given once daily for 5 consecutive days (-/Y + TATk-GFP-CDKL5) (fig. 28C). Scale bar 80 μm. Abbreviations: GR, granular layer; mol, molecular layer.

Fig. 29A-29C show representative images of brain slices displaying treatment of the Dentate Gyrus (DG) molecular layer from: wild type male mice (+/Y) (fig. 29A), CDKL5 knockout male mice (-/Y) (fig. 29B), and CDKL5 knockout male mice treated with TATk-GFP-CDKL 5fusion protein via intraventricular injection given once daily for 5 consecutive days (-/Y + TATk-GFP-CDKL5) (fig. 29C). Scale bar 80 μm. Abbreviations: GR, granular layer; mol, molecular layer.

Figures 30A-30B show graphs showing quantification of synaptic vesicle protein (SYN) optical density in the molecular layer of hippocampus (figure 30A) and the III layer of cortex (figure 30B) in: wild type male mice (+/Y), CDKL5 knockout male mice (-/Y), and CDKL5 knockout male mice treated with TATk-GFP-CDKL 5fusion protein via intraventricular injection given once daily for 5 consecutive days (-/Y + TAT-GFP-CDKL 5). Data are given as fold-differences relative to the corresponding region of the molecular layer or cortex of wild-type mice. Values represent mean ± SD. P < 0.01; p <0.001 compared to +/Y; compared to the-/Y samples, # p <0.05(ANOVA followed by Ponferenb test).

Fig. 31A-31B show graphs demonstrating quantification of optical density of Ser437 phosphorylated-akt (pakt) in molecular layers (fig. 31A) and V layers (fig. 31B) of hippocampus in: wild type male mice (+/Y), CDKL5 knockout male mice (-/Y), and CDKL5 knockout male mice treated with TATk-GFP-CDKL 5fusion protein via intraventricular injection given once daily for 5 consecutive days (-/Y + TATk-GFP-CDKL 5). Data are given as fold-differences relative to the corresponding region of the molecular layer or cortex of wild-type mice. Values represent mean ± SD. P <0.01 compared to +/Y; compared to the-/Y samples, # p <0.01(ANOVA followed by Ponfereny test).

Fig. 32 shows a sketch depicting the implant and fusion protein injection schedules for the behavioral studies illustrated in fig. 33-34.

Fig. 33 shows a graph demonstrating the quantification of the learning period as determined via the Morris Water maze test (Morris Water Mazetest) of: wild-type male mice (+/Y; n ═ 8), CDKL5 knockout male mice (-/Y; n ═ 8), and CDKL5 knockout male mice treated with TATk-GFP-CDKL 5fusion protein (-/Y + TATk-GFP-CDKL 5; n ═ 6). Values represent mean ± SE. P <0.05, P <0.01 compared to untreated wild type case, and # P <0.01 compared to untreated CDKL5 knock out case, as tested with Fisher LSD after ANOVA.

34A-34B show graphs of memory capacity as determined by passive avoidance testing that exhibit: wild-type male mice (+/Y; n ═ 8), CDKL5 knockout male mice (-/Y; n ═ 8), and CDKL5 knockout male mice treated with TATk-GFP-CDKL 5fusion protein (-/Y + TATk-GFP-CDKL 5; n ═ 6). The graph shows the latency to enter the darkened compartment on the first day (fig. 34A) and the second day (fig. 34B) of the behavioral program. Values represent mean ± SE. P <0.001 compared to untreated wild type case and # P <0.01 compared to untreated CDKL5 knock out case, as tested with Fisher LSD after ANOVA.

Figures 35A-35B show graphs showing quantification of locomotor ability as determined by a clasping test in which the total amount of time taken for limbs to clasp during a 2 minute interval was measured as follows: wild-type male mice (+/Y; n ═ 8), CDKL5 knockout male mice (-/Y; n ═ 8), and CDKL5 knockout male mice treated with TATk-GFP-CDKL 5fusion protein according to the injection schedule in figure 32(—/Y + TATk-CDKL 5; n ═ 8). Values represent mean ± SD. P <0.001 compared to +/Y; compared to the-/Y samples, # p <0.001(ANOVA followed by Ponfereny test).

Figure 36 shows body weight (in grams) of wild-type (+/Y) and knockout (/ Y) mice treated with TATk-GFP-CDKL 5fusion protein according to the treatment schedule of figure 20 (+/Y; n ═ 8) or figure 32(—/Y; n ═ 6). After cannulation implantation, mice were allowed to recover for 7 days.

Detailed Description

ATk-CDKL5fusion protein compositions and formulations and methods of their use for treating CDKL 5-mediated diseases and disorders, particularly disorders and diseases caused by mutations and/or defects of CDKL5 are provided herein. Also provided herein are methods for producing TATk-CDKL5fusion protein compositions and formulations. These methods provide improved experimental tools for the study of CDKL 5-mediated neurological disorders and improved treatment options for patients suffering from disorders associated with CDKL5 dysfunction.

Definition of

As used herein, the term "biocompatible" refers to materials, as well as any metabolites or degradation products thereof, that are generally non-toxic to the recipient and do not produce any significant adverse effects on the recipient. Generally, a biocompatible material is one that does not elicit a significant inflammatory or immune response when administered to a patient.

As used herein, the term "molecular weight" generally refers to the mass or average mass of a material. In the case of polymers or oligomers, molecular weight may refer to the relative average chain length or relative chain mass of the bulk polymer. In practice, the molecular weight of polymers and oligomers can be estimated or characterized in different ways, including Gel Permeation Chromatography (GPC) or capillary viscometry. GPC molecular weight is reported as weight average molecular weight (Mw) versus number average molecular weight (Mn). Capillary viscometry provides an estimate of molecular weight as the intrinsic viscosity determined from a diluted polymer solution using a specific set of concentration, temperature and solvent conditions.

As used herein, "biodegradable" generally refers to a material that will degrade or erode under physiological conditions into smaller units or chemicals that can be metabolized, eliminated, or excreted by a subject. Degradation time is a function of composition and morphology. The degradation time may be from hours to weeks.

As used herein, the term "hydrophilic" refers to a substance having a strong polar group that readily interacts with water.

As used herein, the term "hydrophobic" refers to a lack of affinity for water; tend to repel without absorbing water and substances that do not dissolve or mix with water.

As used herein, the term "lipophilic" refers to compounds having affinity for lipids.

As used herein, the term "amphiphilic" refers to molecules that combine hydrophilic and lipophilic (hydrophobic) properties.

As used herein, "about", and the like, when used in conjunction with a numerical variable, generally refer to the value of the variable and to all values of the variable that are within experimental error (e.g., within 95% confidence intervals of the mean) or within +/-10% of the indicated value (whichever is greater).

As used herein, "cell," "cell line," and "cell culture" include progeny. It is also understood that the DNA content of all progeny may not be exactly the same due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological property, as screened for in the originally transformed cell, are included.

As used herein, "composition" refers to a combination of an active agent with at least one other compound or molecule, inert (e.g., a detectable agent or label) or active, such as an adjuvant.

As used herein, a "control" is an alternative subject or sample used for comparative purposes in an experiment and included to minimize or discern the effects of variables other than an independent variable.

As used herein, "positive control" refers to a "control" designed to produce the desired result, provided that all reagents function properly and that the experiment is performed correctly.

As used herein, "negative control" refers to a "control" designed to produce no effect or result, provided that all reagents function properly and that the experiment is performed correctly. Other terms that are interchangeable with "negative control" include "sham treatment (sham)", "placebo (placebo)" and "mock (mock)".

As used herein, "culturing" refers to maintaining cells under conditions in which the cells can multiply and avoid senescence into a population of cells. "culturing" may also include conditions in which the cells are also or alternatively differentiated.

As used herein, "differentially expressed" refers to differentially produced RNA, including but not limited to mRNA, tRNA, miRNA, siRNA, snRNA, and piRNA transcribed from a gene or regulatory region of the genome, or a protein product encoded by a gene, as compared to the level of RNA produced from the same gene or regulatory region in a normal or control cell. In another context, "differentially expressed" also refers to nucleotide sequences or proteins having a different temporal and/or spatial expression profile in a cell or tissue as compared to normal or control cells.

As used herein, "overexpressed" or "overexpression" refers to an increase in the expression level of an RNA or protein product encoded by a gene as compared to the expression level of the RNA or protein product in a normal or control cell.

As used herein, "under-expressed" or "under-expression" refers to a decreased level of expression of an RNA or protein product encoded by a gene as compared to the level of expression of the RNA or protein product in a normal or control cell.

As used herein, an "effective amount" is an amount sufficient to produce a beneficial or desired biological, emotional, medical, or clinical response in a cell, tissue, system, animal, or human. An effective amount may be administered in one or more administrations, administrations or dosages. The term also includes within its scope an amount effective to enhance normal physiological function.

The terms "sufficient" and "effective" as used interchangeably herein refer to an amount (e.g., mass, volume, dose, concentration, and/or time period) necessary to achieve one or more desired results. For example, a therapeutically effective amount refers to the amount required to achieve one or more therapeutic effects.

As used herein, "expansion" or "expanded" in the context of a cell refers to the increase in the number of one or more cell types characteristic of a primary cell population, which may or may not be the same. The original cells used for expansion are not necessarily the same as the cells resulting from expansion. For example, expanded cells may be produced by ex vivo or in vitro growth and differentiation of the original cell population.

As used herein, "expression" refers to the process by which a polynucleotide is transcribed into an RNA transcript. "expression" in the context of mRNA and other translated RNA species refers to one or more processes by which the transcribed RNA is subsequently translated into a peptide, polypeptide, or protein.

As used herein, "isolated" means separated from components, cells, and other materials in which polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof are normally naturally associated. A non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof need not be "isolated" to distinguish it from its naturally occurring counterpart.

As used herein, "concentrated" refers to a molecule, including but not limited to a polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof, that can be distinguished from its naturally occurring counterpart in that the concentration or number/volume of the molecule is greater than the concentration or number/volume of the molecule of its naturally occurring counterpart.

As used herein, "diluted" refers to a molecule, including but not limited to a polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof, that can be distinguished from its naturally occurring counterpart in that the concentration or number/volume of the molecule is less than the concentration or number/volume of the molecule of its naturally occurring counterpart.

As used herein, "isolated" refers to a state that is physically separated from an original source or population such that the isolated compound, agent, particle, or molecule may no longer be considered part of the original source or population.

As used herein, "mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, non-human primates, and zoo, sports, or pet animals such as, but not limited to, dogs, horses, cats, and cattle.

As used interchangeably herein, "subject," "individual," or "patient" refers to a vertebrate organism.

As used herein, "substantially pure cell population" refers to a cell population having the specified cell marker characteristics and differentiation potential that is about 50%, preferably about 75% -80%, more preferably about 85% -90%, and most preferably about 95% of the cells that make up the total cell population. Thus, a "substantially pure population of cells" refers to a population of cells that: under the specified assay conditions, the cell population contains less than about 50%, preferably less than about 20% -25%, more preferably less than about 10% -15%, and most preferably less than about 5% of cells that do not exhibit the specified marker characteristics and differentiation potential.

As used herein, "therapeutic" refers to treating, curing and/or ameliorating a disease, disorder, condition or side effect, or reducing the rate of progression of a disease, disorder, condition or side effect. The term also includes within its scope enhancement of normal physiological function, palliative treatment, and partial remediation of the disease, disorder, condition, side effect, or symptoms thereof. The disease or disorder may be a CDKL5 deficiency and/or Rett syndrome.

As used herein, the terms "treating" and "treatment" generally refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in preventing or partially preventing a disease, symptom or condition thereof, such as a disease or disorder resulting from a mutation and/or defect in CDKL5, CDKL5 variant of Rett syndrome, or other CDKL 5-mediated neurological disorder, and/or may be prophylactic in partially or completely curing the disease, condition, symptom, or adverse effect due to the disease, disorder or condition. As used herein, the term "treatment" encompasses any treatment of a CDKL 5-mediated neurological disorder in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject that may be susceptible to the disease but has not been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) alleviating the disease, i.e., alleviating or ameliorating the disease and/or symptoms or conditions thereof. As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic (preventative) measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

As used herein, "pharmaceutical formulation" refers to the combination of an active agent, compound or ingredient with a pharmaceutically acceptable carrier or excipient, thereby making the composition suitable for diagnostic, therapeutic or prophylactic use in vitro, in vivo or ex vivo.

As used herein, "pharmaceutically acceptable carrier or excipient" refers to a carrier or excipient that can be used to prepare pharmaceutical formulations that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes carriers or excipients that are acceptable for veterinary use as well as human pharmaceutical use. As used in the specification and claims, a "pharmaceutically acceptable carrier or excipient" includes both one and more than one such carrier or excipient.

As used herein, "pharmaceutically acceptable salt" refers to any acid or base addition salt whose counter ion is not toxic to the subject to which they are administered in a pharmaceutical dose of the salt.

As used herein, "preventing" and "prophylaxis" refer to the prevention or termination of a disease or condition before it occurs, even if not diagnosed, or when the disease or condition is still in a subclinical stage.

As used herein, "active agent" or "active ingredient" refers to a substance, compound or molecule that is biologically active or otherwise induces a biological or physiological effect on a subject to which it is administered. In other words, "active agent" or "active ingredient" refers to one or more components of a composition for which all or part of the effect is attributed to the one or more components.

As used herein, "tangible expression medium" refers to a medium that is physically tangible and not merely abstract thinking or unrecorded spoken language. Tangible expression media include, but are not limited to, words on cellulose or plastic materials or data stored on suitable devices such as flash memory or CD-ROM.

As used herein, "chemotherapeutic agent" or "chemotherapeutic agent" refers to a therapeutic agent used to prevent or treat cancer.

As used herein, "matrix" refers to a material in which one or more specialized structures, molecules, or compositions are embedded.

As used herein, "aptamer" refers to a single-stranded DNA or RNA molecule that can bind to a preselected target (including proteins) with high affinity and specificity. The specificity and characteristics of which cannot be determined directly by its primary sequence, but by its tertiary structure.

As used herein, "immunomodulator" refers to an agent, such as a therapeutic agent, which is capable of modulating or modulating one or more immune functions or responses.

As used herein, "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region and a light chain constant region. The VH and VL regions retain binding specificity for antigens and can be further subdivided into hypervariable regions, designated Complementarity Determining Regions (CDRs). The CDRs are interspersed with more conserved regions, designated Framework Regions (FRs). Each VH and VL is composed of three CDRs and four framework regions arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

As used herein, "organism," "host," and "subject" refer to any living entity made up of at least one cell. A living organism can be as simple as, for example, a single isolated eukaryotic cell or cultured cell or cell line, or as complex as a mammal, including humans and animals (e.g., vertebrates, amphibians, fish, mammals, such as cats, dogs, horses, pigs, cows, sheep, rodents, rabbits, squirrels, bears, primates (e.g., chimpanzees, gorillas, and humans)). A "subject" can also be a cell, cell population, tissue, organ, or organism, preferably a human and components thereof.

As used herein, "patient" refers to an organism, host or subject in need of treatment.

As used herein, "protein" as used herein refers to a macromolecule consisting of one or more chains of amino acids in a particular order. The term protein is used interchangeably with "polypeptide". The order is determined by the nucleotide sequence of the nucleotide in the gene encoding the protein. Proteins are required for the structure, function and regulation of cells, tissues and organs of the body. Each protein has a unique function.

As used herein, "substantially pure" means that the target species is the predominant species present (i.e., it is more abundant on a molar basis than any other individual species in the composition), and preferably the substantially purified fraction is a composition in which the target species comprises about 50% of all species present. Generally, a substantially pure composition will comprise greater than about 80%, more preferably greater than about 85%, 90%, 95%, and 99% of all species present in the composition. More preferably, the target species is purified to the necessary homogeneity (contaminant species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single species.

As used herein, "nucleic acid" and "polynucleotide" generally refer to a combined string of at least two base-sugar-phosphates and, in addition, to single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single-and double-stranded RNA, and RNA that is a mixture of single-and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single-and double-stranded regions. Further, a polynucleotide as used herein refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. These regions may comprise all of one or more molecules, but more typically comprise only regions of some molecules. One of the molecules of the triple-helical region is often an oligonucleotide. "Polynucleotide" and "nucleic acid" also encompass such chemically, enzymatically or metabolically modified forms of a polynucleotide, as well as chemical forms of DNA and RNA that are characteristic of viruses and cells, including, inter alia, simple and complex cells. For example, the term polynucleotide includes DNA or RNA containing one or more modified bases as described above. Thus, a DNA or RNA that comprises a rare base (such as inosine) or a modified base (such as a tritylated base) is a polynucleotide as that term is used herein, to name just two examples. "Polynucleotide" and "nucleic acid" also include PNA (peptide nucleic acids), phosphorothioate and other variants of the phosphate backbone of natural nucleic acids. Natural nucleic acids have a phosphate backbone and artificial nucleic acids can contain other types of backbones, but contain the same bases. Thus, a DNA or RNA with a modified backbone for stability or other reasons is a "nucleic acid" or "polynucleotide" as that term is intended herein.

As used herein, "deoxyribonucleic acid (DNA)" and "ribonucleic acid (RNA)" generally refer to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. The RNA may be in the form of: tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), antisense RNA, RNAi (RNA interference construct), siRNA (short interfering RNA) or ribozyme.

As used herein, "nucleic acid sequence" and "oligonucleotide" also encompass nucleic acids and polynucleotides as defined above.

As used herein, "DNA molecule" includes nucleic acids/polynucleotides composed of DNA.

As used herein, "gene" refers to a genetic unit corresponding to a DNA sequence that occupies a particular location on a chromosome and contains genetic instructions for one or more characteristics or one or more traits in an organism.

As used herein, the term "recombinant" generally refers to a non-naturally occurring nucleic acid, nucleic acid construct, or polypeptide. Such non-naturally occurring nucleic acids can include natural nucleic acids that have been modified, e.g., with deletions, substitutions, inversions, insertions, etc., and/or combinations of nucleic acid sequences of different origin joined using molecular biology techniques (e.g., a nucleic acid sequence encoding a fusion protein (e.g., a protein or polypeptide formed from a combination of two different proteins or protein fragments), a nucleic acid encoding a polypeptide in combination with a promoter sequence, wherein the coding sequence and promoter sequence are from different sources or otherwise typically occur together non-naturally (e.g., a nucleic acid and a constitutive promoter), etc.). Recombinant also refers to polypeptides encoded by recombinant nucleic acids. Non-naturally occurring nucleic acids or polypeptides include nucleic acids and polypeptides modified by humans.

As used herein, "fusion protein" refers to a protein formed from a combination of at least two different proteins or protein fragments. The fusion protein is encoded by a recombinant DNA molecule. Thus, a "CDKL 5fusion protein" refers to a recombinant protein having a human CDKL5 polypeptide or variant thereof operably linked to other polypeptide sequences.

As used herein, "CDKL 5 deficient" refers to any deficiency in the biological function of a protein. The defect may result from any DNA mutation in the DNA encoding the protein or in a DNA-associated regulatory region, or any change in protein function due to any change in epigenetic DNA modification, including but not limited to DNA methylation or histone modification, any change in the secondary, tertiary or quaternary structure of the CDKL5 protein, or any change in the ability of the CDKL5 protein to perform its biological function as compared to a wild-type or normal subject.

As used herein, "Rett syndrome variant," "variants of Rett syndrome," and the like refer to atypical forms of Rett syndrome that have similar clinical signs as Rett syndrome but an unknown etiology.

As used herein, "CDKL 5 mutation" refers to any change in the nucleotide sequence of the coding region of the CDKL5 protein.

As used herein, the term "transfection" refers to the introduction of exogenous and/or recombinant nucleic acid sequences into the interior of the cellular membrane enclosed space of a living cell, including the introduction of nucleic acid sequences into the cytosol and the interior space of mitochondria, nuclei or chloroplasts of a cell. The nucleic acid may be in the form of naked DNA or RNA, which may be associated with different proteins or regulatory elements (e.g., promoters and/or signaling elements), or the nucleic acid may be incorporated into a vector or chromosome.

As used herein, "transformation" or "transformed" refers to the introduction of a nucleic acid (e.g., DNA or RNA) into a cell in such a way as to allow expression of the coding portion of the introduced nucleic acid.

As used herein, "transduced" refers to the introduction of a protein directly into a cell.

As used herein, "peptide" refers to a chain of at least 2 amino acids that is short relative to a protein or polypeptide.

As used herein, "variant" refers to a polypeptide that differs from a reference polypeptide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, the differences are limited such that the sequences of the reference polypeptide and the variant are closely similar overall and identical in many regions. The variant and reference polypeptides may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions and/or deletions). The substituted or inserted amino acid residue may or may not be an amino acid residue encoded by the genetic code. Variants of a polypeptide may be naturally occurring such as allelic variants, or may be variants that are not known to be naturally occurring. "variants" include both functional and structural variants.

As used herein, "identity" is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. In the art, "identity" also refers to the degree of sequence relatedness between polypeptides, as determined by the match between such strings of sequences. "consistency" can be readily calculated by known methods including, but not limited to, those described in: computational Molecular Biology (Computational Molecular Biology), Lesk, AM. (Lesk, A.M.), eds, Oxford University Press (Oxford University Press), New York (New York), 1988; biological calculation: informatics and Genome Projects (Biocomputing: information and Genome Projects), Smith, D.W, (Smith, D.W.) eds, Academic Press, new york, 1993; computer Analysis of Sequence Data (Computer Analysis of Sequence Data), part I, Griffin, A.M, (Griffin, A.M.) and Griffin, h.g., editions, limana Press (Humana Press), New Jersey, 1994; sequence Analysis in Molecular Biology (Sequence Analysis in Molecular Biology), von hydantoin cutter, g. (von heinje, G.), editors, academic press, 1987; and Sequence analysis primary reads (Sequence analysis primer), gibbocov, M. (Gribskov, M.) and defuzk J. (Devereux, J.), eds, michelson Press (M Stockton Press), new york, 1991; and carriro, h. (Carillo, H.) and riemann, d. (Lipman, D.), 1988, 48:1073, journal of Applied Math of the institute of industrial and Applied mathematics (SIAM J Applied Math). A preferred method for determining identity is designed to yield the maximum match between test sequences. Methods of determining consistency are codified in publicly available computer programs. The percent identity between two sequences can be determined by using Analysis Software (e.g., the Sequence Analysis Software Package of the genetic computing Group (Sequence Analysis Software Package of the genetics Computer Group), Madison Wis., Wis.) in conjunction with the Needman and Wunsch (J.mol.biol.), 1970, 48: 443) algorithms (e.g., NBLAST and XBLAST) of Needman and Wunsch (J.mol.biol.), Needman and Wunsch). Default parameters are used to determine the identity of the polypeptides of the disclosure.

As used herein, "plasmid" as used herein refers to a non-chromosomal double-stranded DNA sequence comprising an intact "replicon", thereby allowing the plasmid to be replicated in a host cell.

As used herein, the term "vector" is used to refer to a vehicle for introducing an exogenous nucleic acid sequence into a cell. A vector may comprise a linear or circular DNA molecule (e.g., a plasmid) comprising a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription and translation upon introduction into a host cell or host cell organelle. Such additional segments may include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are typically derived from yeast or bacterial genomes or plasmid DNA, or viral DNA, or may contain elements of both.

As used herein, "operably linked" indicates that the regulatory sequences useful for expressing the coding sequence of a nucleic acid are positioned in the nucleic acid molecule at the appropriate location relative to the coding sequence in order to effect expression of the coding sequence. This same definition sometimes applies to the arrangement of coding sequences and transcriptional control elements (e.g., promoter, enhancer, and termination elements) and/or selectable markers in an expression vector.

As used herein, "wild-type" is the typical form of an organism, variety, strain, gene, protein or characteristic that occurs in nature, as distinguished from variant forms that may arise from selective breeding or transformation using transgenes.

As used herein, "cDNA" refers to a DNA sequence that is complementary to an RNA transcript in a cell. It is an artificial molecule. Typically, cDNA is formed in vitro by an enzyme called reverse transcriptase using RNA transcripts as templates.

As used herein, "purified" or "purification" is used to refer to a nucleic acid sequence, peptide, or polypeptide having increased purity relative to the natural environment.

As used herein, "differentiation" or "differentiation" refers to the process by which precursor or progenitor cells (e.g., neuronal progenitor cells) differentiate into a particular cell type (e.g., neurons).

As used herein, "dose", "unit dose" or "dose" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined amount of a CDKL5fusion protein, a composition containing a CDKL5fusion protein, and/or a pharmaceutical formulation thereof, calculated to produce a desired response or a response associated with administration thereof.

As used herein, a "specific binding partner" or "binding partner" is a compound or molecule that binds with a second compound or molecule with a higher affinity than all other molecules or compounds.

As used herein, "specifically binds" or "specifically binds" refers to the binding that exists between such paired species such as enzymes/substrates, receptors/agonists or antagonists, antibodies/antigens, lectins/carbohydrates, oligo-DNA primers/DNA, enzymes or proteins/DNA, and/or RNA molecules and other nucleic acids (DNA or RNA) or amino acids, which binding may be mediated by covalent interactions or non-covalent interactions or a combination of covalent and non-covalent interactions. When the interaction of two species produces a non-covalently bound complex, the binding that occurs is typically the result of electrostatic, hydrogen bonding, or lipophilic interactions. Thus, "specific binding" occurs between pairs of species, where there is an interaction between the two that results in a binding complex with the following characteristics: antibody/antigen, enzyme/substrate, DNA/DNA, DNA/RNA, DNA/protein, RNA/amino acid, receptor/substrate interactions. In particular, specific binding is characterized by one member of the pair binding to a particular species within the family of compounds to which the corresponding member of the binding member belongs and not to other species. Thus, for example, an antibody preferably binds to a single epitope within a protein family and does not bind to other epitopes.

As used herein, "anti-infective" refers to a compound or molecule that can kill or inhibit the spread of an infectious agent. Anti-infective agents include, but are not limited to, antibiotics, antibacterial agents, antifungal agents, antiviral agents, and antiprotozoal agents.

As used herein, "wild-type" is the typical form of an organism, variety, strain, gene, protein or characteristic that occurs in nature, as distinguished from variant forms that may arise from selective breeding or transformation using transgenes.

As used herein, "induce", "inducing" or "induced" refers to activating or stimulating processes or pathways within a cell, such as endocytosis, secretion and exocytosis.

As used herein, "derivative" refers to any compound having the same or similar core structure as the compound, but having at least one structural difference including substitution, deletion, and/or addition of one or more atoms or functional groups. The term "derivative" does not mean that the derivative is synthesized from the parent compound as a starting material or intermediate, but may be the case. The term "derivative" may include a prodrug, or a metabolite of the parent compound. Derivatives include those in which the free amino group in the parent compound has been derivatized to form an amine salt of hydrochloric acid, p-toluenesulfonamide, benzoyloxycarboxamide (benzoxycarboxamides), t-butoxycarboxamides (t-butyloxycarocarboxamides), thiocarbamate-type derivatives, trifluoroacetamide (trifluoroacetamides), chloroacetamide or formamide. Derivatives include compounds in which the carboxyl groups in the parent compound have been derivatized to form methyl and ethyl esters, or other types of esters or hydrazides. Derivatives include compounds in which the hydroxyl groups in the parent compound have been derivatized to form O-acyl or O-alkyl derivatives. Derivatives include compounds in which the hydrogen-donating bond group in the parent compound is replaced with another hydrogen-donating bond group such as OH, NH, or SH. Derivatives include the replacement of a hydrogen bond accepting group in the parent compound with another hydrogen bond accepting group such as esters, ethers, ketones, carbonates, tertiary amines, imines, thiones, sulfones, tertiary amides, and sulfides. "derivatives" also include the replacement of the extension of the cyclopentane ring by saturated or unsaturated cyclohexane or other more complex, e.g. nitrogen containing, rings and extensions of these rings with different side groups.

As used herein, "therapeutically effective amount" refers to the amount of a CDKL5fusion protein, a composition containing a CDKL5fusion protein, a pharmaceutical formulation thereof, an adjuvant, or a second agent described herein that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. A "therapeutically effective amount" includes an amount of a CDKL5fusion protein, a composition containing a CDKL5fusion protein, a pharmaceutical formulation thereof sufficient to prevent, reduce or alleviate to some extent one or more symptoms of a CDKL5 deficiency and/or Rett syndrome when administered alone or in conjunction with a second agent. A "therapeutically effective amount" includes an amount of a CDKL5fusion protein, a composition containing a CDKL5fusion protein, a pharmaceutical formulation thereof sufficient to increase neuron survival, neuron number, neurite outgrowth, elongation and/or branch density in a region of the brain of a subject, as compared to a control, when administered alone or co-administered with a second agent. A "therapeutically effective amount" includes an amount of a CDKL5fusion protein, a composition containing a CDKL5fusion protein, a pharmaceutical formulation thereof, sufficient to increase learning ability of a subject, as compared to a control, when administered alone or co-administered with a second agent. A "therapeutically effective amount" includes an amount of a CDKL5fusion protein, a composition containing a CDKL5fusion protein, a pharmaceutical formulation thereof, sufficient to increase the memory capacity of a subject, as compared to a control, when administered alone or in combination with a second agent. A "therapeutically effective amount" includes an amount of a CDKL5fusion protein, a composition containing a CDKL5fusion protein, a pharmaceutical formulation thereof, sufficient to improve motor function in a subject, as compared to a control, when administered alone or in combination with a second agent. A "therapeutically effective amount" includes an amount of a CDKL5fusion protein, a composition containing a CDKL5fusion protein, pharmaceutical formulations thereof, sufficient to restore learning, memory, and/or motor function to a level substantially similar to wild-type or normal levels when administered alone or in conjunction with a second agent. "therapeutically effective amount" includes an amount of a CDKL5fusion protein, a composition containing a CDKL5fusion protein, a pharmaceutical formulation thereof, which is sufficient to restore neuron number, neuron survival, neurite growth, neurite elongation, neurite branch number, and/or neurite branch density in a region of the brain to a level substantially similar to wild-type or normal levels, when administered alone or in combination with a second agent. The therapeutically effective amount will vary according to: CDKL5fusion protein, compositions containing CDKL5fusion protein, exact chemical structure of its pharmaceutical formulation, CDKL5 deficiency being treated, Rett syndrome or symptoms thereof, route of administration, time of administration, rate of excretion, drug combination, judgment of the treating physician, dosage form, and age, weight, general health, gender, and/or diet of the subject to be treated.

As used herein, "synergistic effect," "synergy," or "synergy" refers to an effect that occurs between two or more molecules, compounds, substances, factors, or compositions that is greater than or different from the sum of their individual effects.

As used herein, "additive effect" refers to an effect that is equal to or the same as the sum of their individual effects occurring between two or more molecules, compounds, substances, factors or compositions.

Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Discussion of the related Art

TATk-CDKL5fusion gene and protein

Fusion gene and protein

Disclosed herein are recombinant cDNA sequences encoding different CDKL5fusion proteins containing modified tat (tatk) sequences. In one embodiment, the fusion protein contains a human CDKL5 polypeptide operably linked to a TATk polypeptide. The cDNA sequence encoding the CDKL5fusion protein can have a sequence according to any one of SEQ ID NOs 2, 7, 9, 11, 13, or variants thereof, described herein. The CDKL5fusion protein may have a polypeptide sequence according to any one of SEQ ID NOs 8, 10, 12, 14, or variants thereof, described herein.

In some embodiments, the human CDKL5cDNA sequence may be according to SEQ ID NOs 1 or 15. In further embodiments, the human CDKL5cDNA may have from about 90% to about 100%, 80% to about 90%, or about 50% to about 80% identity to SEQ ID No. 1 or 15. In some embodiments, the human CDKL5cDNA sequence may encode an amino acid sequence according to SEQ ID NOs 2 or 16. In further embodiments, the human CDKL5cDNA sequence may encode an amino acid sequence having from about 90% to about 100%, 80% to about 90%, or about 50% to about 80% identity to SEQ ID No. 2 or 16.

In some embodiments, the human CDKL5cDNA sequence may be a fragment of at least 12 consecutive nucleotides having about 90% to 100% identity to 12 consecutive nucleotides in SEQ ID NO: 1. In some embodiments, the human CDKL5cDNA sequence may be a fragment of at least 12 consecutive nucleotides having about 80% to 90% identity to 12 consecutive nucleotides in SEQ ID NO: 1. In some embodiments, the cDNA sequence may be a fragment of at least 12 contiguous nucleotides with about 50% to 80% identity to 12 contiguous nucleotides in SEQ ID NO: 1.

The CDKL5fusion protein contains a modified trans-acting activated transcription (TAT) Protein Transduction Domain (PTD) (hereinafter TATk) operably linked to a human CDKL5 polypeptide. TATk can have a cDNA sequence according to SEQ ID NO. 3 and an amino acid sequence according to SEQ ID NO. 4. TATk is a modified TAT-PTD. Unmodified TAT-PTD mediates transduction of peptides and proteins into cells. However, unmodified TAT-PTD does not allow secretion of the TAT-PTD fusion protein by cells. The unmodified TAT-PTD is cleaved from the fusion protein by furin cleavage protease at a furin recognition sequence located within the unmodified TAT-PTD. In contrast, TATk is modified such that it does not contain a furin recognition sequence. Thus, the CDKL5fusion proteins described herein that contain TATk can be secreted by eukaryotic cells in their full-length form.

In some embodiments, the TATk cDNA sequence may be about 90% to 100% or about 80% to about 90% identical to SEQ ID No. 3. In some embodiments, the TATk cDNA may encode a polypeptide sequence having about 90% to 100% or about 80% to about 90% identity to SEQ ID No. 4.

The CDKL5fusion protein may optionally contain an Ig kappa-chain leader sequence to direct the polypeptide to the secretory pathway during production by a cell. In some embodiments, the Ig kappa-chain leader sequence may be operably linked at the N-terminus of a human CDKL5 polypeptide. The Ig kappa-chain leader sequence may have a cDNA sequence according to SEQ ID No. 5 or variants thereof described herein and may have an amino acid sequence according to SEQ ID No. 6 or variants thereof described herein.

In other embodiments, the Ig kappa-chain leader cDNA may be about 90% to 100%, about 80% to about 90%, or about 80% to 90% identical to SEQ ID No. 5. In some embodiments, the Ig kappa-chain leader sequence may have an amino acid sequence that is about 90% to about 100%, about 80% to about 90%, or about 50% to about 80% identical to SEQ ID No. 6.

The CDKL5fusion protein may optionally contain one or more protein tags operably linked to a CDKL5fusion protein. These tag types are amino acid sequences that allow for affinity purification, solubilization, chromatographic separation, and/or immunodetection of the fusion protein. Suitable protein tags include, but are not limited to, Chitin Binding Protein (CBP), Maltose Binding Protein (MBP), glutathione-S-transferase (GST), poly (His), Thioredoxin (TRX), poly (NANP), FLAG tag (including any FLAG tag variant, e.g., 3x FLAG), V5 tag, Myc tag, HA tag, S tag, SBP tag, Sf tag 1, Sof tag 3, Tc tag, Xpress tag, Strep tag, Isopep tag, Spy tag, Ty tag, Biotin Carboxyl Carrier Protein (BCCP), and Nus tag. The CDKL5fusion protein cDNA according to SEQ ID NO 7, 9 or 11 having the amino acid sequence according to SEQ ID NO 8, 10 or 12, respectively, shows a non-limiting example of a CDKL5fusion protein comprising a TATk and Myc tag and a poly (HIS) tag. The CDKL5fusion protein cDNA according to SEQ ID NO 13, having the amino acid sequence according to SEQ ID NO 14, shows a non-limiting example of a CDKL5fusion protein with a FLAG tag.

The CDKL5fusion protein may optionally contain one or more reporter proteins operably linked to a CDKL5 polypeptide. Suitable reporter genes include, but are not limited to, fluorescent proteins (e.g., Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP), Yellow Fluorescent Protein (YFP), Blue Fluorescent Protein (BFP), and Cyan Fluorescent Protein (CFP)), β -galactosidase, luciferase (bacterial, firefly, and renilla luciferase), antibiotic resistance genes (e.g., chloramphenicol acetyltransferase, neomycin phosphotransferase, and NPT-II), β -glucuronidase (p-glucuronidase), and alkaline phosphatase. The inclusion of a reporter protein allows, inter alia, direct and/or indirect characterization of the fusion protein and the function of the fusion protein, as well as affinity purification of the protein. The reporter protein may be operably linked to the N-terminus and/or C-terminus of the human CDKL5 polypeptide. In other embodiments, the reporter protein may be operably linked to the N-terminus and/or C-terminus of the CDKL5fusion protein. The CDKL5fusion protein cDNA according to SEQ ID NO 9 or 11 and having the amino acid sequence according to SEQ ID NO 8 or 10, respectively, shows a non-limiting example of a CDKL5fusion protein containing a fluorescent reporter protein.

Recombinant vector

The CDKL5fusion cDNA sequence may be incorporated into a suitable expression vector. The expression vector may contain one or more regulatory sequences or one or more other sequences for facilitating expression of the CDKL5fusion cDNA. The expression vector may contain one or more regulatory sequences or one or more other sequences for facilitating replication of the CDKL5fusion expression vector. The expression vector may be suitable for expressing the CDKL5fusion protein in bacterial cells. In other embodiments, the expression vector may be suitable for expressing the CDKL5fusion protein in a yeast cell. In further embodiments, the expression vector may be suitable for expressing the CDKL5fusion protein in a plant cell. In other embodiments, the expression vector may be suitable for expressing a CDKL5fusion protein in a mammalian cell. In another example, the vector may be suitable for expressing a CDKL5fusion protein in a fungal cell. Suitable expression vectors are generally known in the art.

TATk-CDKL5 protein production

In some embodiments, the CDKL5fusion protein is produced in vitro in a cell culture system. The cell culture system may contain one or more bacterial, yeast, fungal, plant or mammalian cells. In some embodiments, the CDKL5fusion protein is secreted into the cell culture medium by one or more cultured cells. In other embodiments, the CDKL5fusion protein is contained within the cytoplasm or cell membrane of one or more cultured cells.

Thus, turning to fig. 1, one embodiment of a method of producing a CDKL5fusion protein is shown, wherein a CDKL5fusion protein is produced by a cultured cell and secreted into the surrounding culture medium. The method begins by transfecting or otherwise delivering a suitable vector containing the CDKL5fusion protein cDNA sequence into one or more cells in culture (6000). The cells (6010) are then cultured using commonly known methods such that the transfected cells produce the CDKL5fusion protein from the vector and secrete the CDKL5fusion protein into the surrounding cell culture medium. After an appropriate amount of time, the medium containing the secreted CDKL5fusion protein was collected (6020). In some embodiments, the cells are cultured for from about 12h to about 96 h. At this time, it was determined whether further purification of the CDKL5fusion protein from the culture medium was required (6030). In some embodiments, the media containing the CDKL5fusion protein is not further purified and is used directly to transduce one or more cells (6050). In other embodiments, the CDKL5fusion protein is further purified from the culture medium and/or the CDKL5fusion protein is concentrated in the culture medium. In some embodiments, the CDKL5fusion protein is purified and/or concentrated using a suitable method. Suitable methods include, but are not limited to, affinity purification, size exclusion separation, and chromatographic separation methods.

Turning to fig. 2, which illustrates one example of a method of producing a CDKL5fusion protein, wherein the CDKL5fusion protein is not secreted into the surrounding cell culture medium, in view of the understanding of the secretory production method. The method begins by transfecting or otherwise delivering a suitable vector containing the CDKL5fusion protein cDNA sequence into one or more cells in culture (6000). The cells (6010) were then cultured using commonly known methods to allow the transfected cells to produce the CDKL5fusion protein from the vector. After an appropriate amount of time, the cells were lysed using standard methods (7000). In some embodiments, the cells are cultured for from 12h to 96h prior to lysis.

Next, it was determined whether the CDKL5fusion protein was integrated within the cell membrane or cytoplasm (7010). If the CDKL5fusion protein is in the cell membrane fraction, the cell membrane fraction is collected (7020). After collection of the cell membrane fraction (7020), the CDKL5fusion protein is separated from the membrane fraction using a suitable method (6040) for purification and/or concentration of the CDKL5fusion protein.

In the example where the CDKL5fusion protein is present in the cytoplasm, the supernatant containing the CDKL5fusion protein was collected (7030). After collecting the supernatant (7030), it was determined whether the CDKL5fusion protein should be further purified and/or concentrated. If it is determined that the CDKL5fusion protein should be further purified and/or concentrated, the CDKL5fusion protein is purified and/or concentrated using a suitable method (6040). Suitable methods include, but are not limited to, affinity purification, size exclusion separation, and chromatographic separation methods. In other embodiments where it is determined that CDKL5 should not be further purified and/or concentrated from the supernatant, the supernatant containing the CDKL5fusion protein is used directly to transduce cells (6050).

Compositions and formulations containing TATk-CDKL5fusion protein

Compositions and formulations containing the CDKL5fusion protein as described herein are also within the scope of the present disclosure. The composition can be a culture medium or supernatant containing a CDKL5fusion protein that can be produced according to the methods described herein.

The CDKL5fusion proteins described herein can be provided to a subject in need thereof, either alone or as an active ingredient, such as in a pharmaceutical formulation. Thus, also described herein are pharmaceutical formulations containing an amount of CDKL5fusion protein. In some embodiments, the pharmaceutical formulations contain a therapeutically effective amount of a CDKL5fusion protein. The pharmaceutical formulations described herein can be administered to a subject in need thereof. The subject in need thereof may have a CDKL5 deficiency, Rett syndrome, and/or symptoms thereof. In other embodiments, the CDKL5fusion protein may be used in the manufacture of a medicament for treating or preventing a CDKL5 deficiency, Rett syndrome, and/or symptoms thereof.

Pharmaceutically acceptable carriers and auxiliary ingredients and reagents

Pharmaceutical formulations containing a therapeutically effective amount of the CDKL5fusion proteins described herein may further comprise a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oils, fatty acid esters, hydroxymethyl cellulose, and polyvinylpyrrolidone, which do not deleteriously react with the active composition.

These pharmaceutical formulations can be sterilized and, if desired, mixed with auxiliaries, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring substances, aromatic substances and/or fragrances and the like, which do not deleteriously react with the active composition.

In addition to a therapeutically effective amount of the CDKL5fusion protein described herein, the pharmaceutical formulation may also contain an effective amount of an auxiliary active agent, including, but not limited to, DNA, RNA, amino acids, peptides, polypeptides, antibodies, aptamers, ribonucleases, ribonuclease guide sequences, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatory agents, antihistamines, anti-infective agents, and chemotherapeutic agents that inhibit the translation or transcription of essential tumor proteins and genes.

Suitable hormones include, but are not limited to, amino acid derived hormones (e.g., melatonin and thyroxine), small peptide and protein hormones (e.g., thyroid stimulating hormone releasing hormone, antidiuretic hormone, insulin, growth hormone, luteinizing hormone, follicle stimulating hormone, and thyroid stimulating hormone), eicosanoids (eicosanoids) (e.g., arachidic acid, lipoxin, and prostaglandins), and steroid hormones (e.g., estradiol, testosterone, tetrahydrotestosterone cortisol).

Suitable immunomodulators include, but are not limited to, prednisone, azathioprine, 6-MP, cyclosporine, tacrolimus, methotrexate, interleukins (e.g., IL-2, IL-7, and IL-12), cytokines (e.g., interferons (e.g., IFN- α, IFN- β, IFN- κ, IFN- ω, and IFN- γ), granulocyte colony stimulating factor, and imiquimod), chemokines (e.g., CCL3, CCL26, and CXCL7), cytosine phosphate-guanosine, oligodeoxynucleotides, dextran, antibodies, and aptamers).

Suitable antipyretics include, but are not limited to, non-steroidal anti-inflammatory drugs (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), aspirin and related salicylates (e.g., choline salicylate, magnesium salicylate (magnesium salicylate), and sodium salicylate (sodium salicylate)), acetaminophen (paracetamol)/acetaminophen (acetaminophen), metamizazole, nabumetone, antipyrine, and quinine.

Suitable anxiolytics include, but are not limited to, benzodiazepines (e.g., alprazolam, bromocriptan, lisinopril, clonazepam, chlordiazepam, diazepam, flurazepam, lorazepam, oxazepam, temazepam, triazolam and tofisopam), 5-hydroxytryptamine antidepressants (e.g., selective 5-hydroxytryptamine reuptake inhibitors, tricyclic antidepressants and monoamine oxidase inhibitors), merbica (mebicar), afobazole, selank, bromantane (bromantane), 3-hydroxy-6-methyl-2-ethyl pyridine hydrochloride (emoxypine), azaperone (apizarons), barbiturates, hydroxyzine, pregabalin, valdolol, and beta blockers.

Suitable antipsychotic agents include, but are not limited to, benproperidol (benproperidol), bromperidol (bromoperidol), haloperidol (haloperidol), moperone (moperone), pipamperone (piperone), timiperone, fluspirilene, penfluridol, pimozide (pimozide), acepromazine, chlorpromazine, cyameprazine, dizyrazine, fluphenazine, levopromazine, thiamphenzine, perazine (perazine), piperazine, perphenazine, pipothiazine, prochlorperazine, promazine (promazine), promazine, prothioxazine, prothioconazole (thioridazine), trifluoperazine, triprolidine, chlorprothixene, thiothixene, thioperazine (thioproperitone), thioperazine (thioperazine), thioperazine (topride), topride (topride, topride (topride), topride (topride), topride (topride ), topride (topride, topride, Verapride (veralipride), amisulpride (amisulpride), amoxapine (amoxapine), aripiprazole, asenapine (asenapine), clozapine (clozapine), blonanserin (blonanserin), ilone (iloperidone), lurasidone (lurasidone), melperone (melperone), nemobipride (nemonapride), olanzapine (nzapine), paliperidone (paliperidone), piropiropirone (peroxolone), quetiapine (quetiapine), remoxipride (remoxipride), risperidone (risperidone), sertindole (sertindole), trimipramine, ziprasidone, zotepine (zotepine), alstonie, percentilex (befelone), bitorubin (piroctone), penoxepidine (penoxepidine), penoxepirubine (clavine), penoxepidine (penoxepidine), penoxepirubine (penoxepirubine), penoxepirubine (penoxepidine), penoxepidine (penoxepirubine (s (penoxepidine), and penoxepirubine (penoxepidine).

Suitable analgesics include, but are not limited to, acetaminophen/acetaminophen, non-steroidal anti-inflammatory drugs (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), opioids (e.g., morphine, codeine, oxycodone, hydrocodone, dihydromorphine, meperidine, buprenorphine), tramadol (tramadol), norepinephrine, flupirtine, nefopam, oxfenadrine, pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone, ketonide, piperazinemide, and aspirin, as well as related salicylates (e.g., choline salicylate, magnesium salicylate, and sodium salicylate).

Suitable antispasmodics include, but are not limited to, mebeverine, papaverine (papverine), cyclobenzaprine, carisoprodol, oxfenadrin, tizanidine, metaxalone, methodcarbamol (methodcarbamol), chlorzoxazone, baclofen, dantrolene (dantrolene), baclofen, tizanidine, and dantrolene.

Suitable anti-inflammatory agents include, but are not limited to, prednisone, non-steroidal anti-inflammatory drugs (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), and immunoselective anti-inflammatory derivatives (e.g., submandibular peptide-T and derivatives thereof).

Suitable antihistamines include, but are not limited to, H1-receptor antagonists (e.g., acrivastine, azelastine (azelastine), bilastine (bilastine), brompheniramine, buclizine, brompheniramine, carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine, clemastine (clematine), cyproheptadine, desloratadine, dexbrompheniramine (dexbrompheniramine), dexchlorpheniramine, dimenhydrinate, diphenhydramine, diammine (doxylamine), ebastine (ebasine), enbranamine (embramine), fexofenadine, hydroxyzine, levocetirizine (levocetirizine), loratadine (loratadine), meloxicam, milnacipramine, olazine, oxfenfenadine, pheniramine (e), trimetrezine (84), pyrilamine (e), trimethoprim, Famotidine, lafutidine, nizatidine (nizatidine), ranitidine (rafatidine) and roxatidine (roxatidine)), tritoquinoline, catechin (catehin), cromoglycate, nedocromil, and beta 2-adrenergic agonists.

Suitable anti-infective agents include, but are not limited to, anti-amebiases (nitazoxanide, paromomycin, metronidazole, tinidazole, chloroquine, miltefosine (miltefosine), amphotericin b, and diiodoquine), aminoglycosides (e.g., paromomycin, tobramycin, gentamicin, amikacin (amikacin), kanamycin, and neomycin), anthelmintics (e.g., pyrantel, mebendazole, ivermectin), praziquantel, abendazole (abendazole), thiabendazole, oxanquin), antifungals (e.g., azole antifungals (e.g., itraconazole), fluconazole, posaconazole, ketoconazole, clotrimazole, miconazole, and voriconazole), echinocandins (e.g., caspofungin, anidulafungin, and micafungin (micafungin)), griseofulvin, terbinafine (terbinafine), cytosines (e.g., amphotericin, and gentin (e, betahistidins), and fungin (e.g., amphotericin), fungin (e.g., fungicins, and gentamikacin (e., Antimalarial agents (e.g. pyrimethamine/sulfadoxine), artemether/lumefantrine, atovaquone/proguanil, quinine, hydroxychloroquine, mefloquine, chloroquine, doxycycline, pyrimethamine and halofantrine), antineoplastics (e.g. aminosalicylates, isoniazid/rifampin, isoniazid/pyrazinamide/rifampin, bedaquiline, isoniazid, ethambutol, rifampin, rifabutin, capreomycin and cycloserine), antiviral agents (e.g. amantadine, rimantadine, abacavir (abacavir)/lamivudine, emtricitabine/tenofovir), tenofovir/tenofovir (tenofovir)/ranitidine (tenofovir), and the like, Efavirenz/emtricitabine/tenofovir, abacavir (avavir)/lamivudine/zidovudine (zidovudine), lamivudine/zidovudine, emtricitabine/tenofovir, emtricitabine/lopinavir (opinavir)/ritonavir (ritonavir)/tenofovir, interferon alpha 2 v/ribavirin (ribavirin), peginterferon alpha-2 b, maraviroc (maraviroc), raltegravir (raltegravir), deroglivir (doltegravivir), envirvir (enfuvirtide), foscarnet, fomivirsen (mipivirsen), oseltamivir (oseltamivir), zanavir (zanavir), nevirapine (nevirapine), efavirenz, riline, tenofovir (rilividine), abacavir, rilividine, adefovir, tenofovir, rilividine, tenofovir, rilividine, rilevirine, rilivir, rilevirine, rilevine, nevirapine, avacivr, zidovudine, stavudine, emtricitabine, zalcitabine (xalitabine), tipifdine, cimetimivir, boceprevir, telaprevir, lopinavir/ritonavir, fosamprenvir (fosamprenvir), rinnavir (dranuavir), ritonavir, tipranavir (tipranavir), atazanavir (atazanavir), nelfinavir (nelavir), amprenavir, indinavir, saquinavir (sawinuavir), ribavirin, valacyclovir (valcyclovvir), acyclovir, famciclovir, ganciclovir and valganciclovir (valganciclovir)), carbapenems (e.g. doripenem, meropenem, etanan and cetin/pezil), cephalosporins (e.g. doxepinasil, ceftiovir, ceftriaxone, ceft, Chlorocarbon, cefoxitin, cefaclor, ceftibuten, ceftriaxone, cefotaxime (cefotaxime), cefpodoxime, cefdinir, cefixime, cefditoren, cefazolin (cefixime), and ceftazidime), glycopeptide antibiotics (e.g., vancomycin, dalbavancin, oritavancin, and telavancin (telvencin)), glycinyclines (e.g., tigecycline), antihyperglycemics (e.g., clofazimine and salaminoxidin), lincomycin (lincomycin) and derivatives thereof (e.g., clindamycin and lincomycin), macrolides and derivatives thereof (e.g., telithromycin, fidaxomicin), erythromycin (azithromycin), azithromycin, clarithromycin, dirithromycin, and oleandomycin), linezolid (linezolizolid), sulfamethoxazole/trimethoprim, rifaximin, chloramphenicol, fosfomycin, metronidazole, amitrazol (aztream), ceftriam (ceftriaxone), ceftriaxone, ceftizom, Bacitracin, penicillins (penicilins) (amoxicillin, ampicillin, bacampicillin, carbenicillin, piperacillin, ticarcillin, amoxicillin/clavulanic acid, ampicillin/sulbactam, piperacillin/tazobactam, clavulanic acid/ticarcillin, penicillins, procainazine (procaine penillin), oxacillin (oxaxilin), dicloxacillin and nafcillin), quinolones (e.g., lomefloxacin, norfloxacin, ofloxacin, qatifloxacin, moxifloxacin, ciprofloxacin, levofloxacin, gemifloxacin, moxifloxacin, cinoxacin, nalidixic acid, enoxacin (enoxacin), glafloxacin, gatifloxacin (gatifloxacin), trovafloxacin and sparfloxacin), sulfonamides (e.g., sulfamethoxazole/trimethoprim, sulfasalazine and sulfamethoxazole (sulfamethoxazole)) (sulfamethoxazole), Tetracyclines (e.g., doxycycline, demeclocycline, minocycline, doxycycline/salicylic acid, doxycycline/omega-3 polyunsaturated fatty acids, and tetracycline) and urinary tract anti-infectives (e.g., nitrofurantoin, urotropin, fosfomycin, cinoxacin, nalidixic acid, trimethoprim, and methylene blue).

Suitable chemical agents include, but are not limited to, paclitaxel, berentuzumab (brentuximabvedotin), doxorubicin, 5-FU (fluorouracil), everolimus, pemetrexed, melphalan, pamidronate sodium, anastrozole, exemestane, nelarabine, efavirenzumab, bevacizumab, belinostat (belinostat), tositumomab, carmustine, bleomycin, bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib (vandetanib), bicalutamide, lomustine, daunorubicin, clofarabine, cabozantinib, dactinomycin, ranibixemab, ramucirumab (ramucirumab), arabinoside, pratensacin (cytoxan), cyclophosphamide (cyclophophamide), decitabine, dexamethasone, docetaxel, hydroxyurea, amiloride, leuprolide, oxaliplatin, aspartamicin, estramustine, sultamide, etc, Vismodegib (vismodegib), Asampsin Erwinia chrysanthemi (aspraginase Erwinia chrysanthemi), amifostine, etoposide, flutamide, toremifene, fulvestrant, letrozole, degarelix (degarelix), pralatrexate, methotrexate, floxuridine, obinutuzumab, gemcitabine, afatinib, imatinib mesylate (imatinib mesylate), carmustine, eribulin (eribulin), trastuzumab, altretamine, topotecan, panatinib, idarubicin, ifosfamide, ibrutinib, acitinib, interferon alpha-2 a, gefitinib, romidepsin (roepimsin), ixabepilone, casuaritinib, cabazitaxel, adaptotemab, dol-tranemin, carfilzomib, chlorambucil, gentamifostine, vone, vozine, vincristine (vincristine), vinorelbine, trogliptin (vincristine), vinorelbine, tretinomycin, vindol, tretinomycin, tremulin (vinpocetine), tremulin, vinpocetine, tremulin, tremulins, tremulin, tremulins, tremul, Mesna (mesna), strontium-89 chloride, mechlorethamine (meclorethamine), mitomycin, busulfan, gemtuzumab ozogamicin, vinorelbine, filgrastim (filgrastim), pegylated filgrastim, sorafenib, nilutamide, pentostatin, tamoxifen, mitoxantrone, pemetrexed, dinetonase, dinebukin-toxin linker (denileukin diftitox), alitretinoin, carboplatin, pertuzumab, cisplatin, pomalidomide (pomidomide), prednisone, aldesleukin, mercaptopurine, zoledronic acid, lenalidomide, rituximab, octreotide (octrotide), dasatinib, regorafenib, histrelin, sunitinib, siuximab (siuximab), omaine (omaxipine), thioguanine (omaxilaxoline), thioguanine (thioguanine), thiotepine (tiazotifloxapine), thiotepine (tiadinine), thiotepenine (tiadinine, tiadinine (tiadinine), thiotepine (tiadinine), neratidine (tiadinine, tiadinine (tiadinine), tiadinine (tiadinine), tiadinine (tiadinine, BCG, sirolimus (temsirolimus), bendamustine hydrochloride, triptorelin, arsenic trioxide, lapatinib, valrubicin (valrubicin), panitumumab, vinblastine, bortezomib, tretinoin, azacitidine, pazopanib, teniposide, folinic acid, crizotinib (crizotinib), capecitabine, enzalutamide (enzalutamide), ipilimumab, goserelin, vorinostat (vorinostat), idalisib (idelisib), ceritinib (ceritinib), abiraterone, epothilone, tafluoroperioside (tafluoside), azathioprine, floxuridine, vindesine, and all-trans retinoic acid.

Effective amounts of CDKL5fusion protein and adjuvant

The pharmaceutical formulation may contain a therapeutically effective amount of the CDKL5fusion protein, and optionally a therapeutically effective amount of an adjuvant. In some embodiments, a therapeutically effective amount of a CDKL5fusion protein may range from about 1 μ g/kg to about 10 mg/kg. In further embodiments, a therapeutically effective amount of a CDKL5fusion protein may range from about 1ng/g body weight to about 0.1mg/g body weight. A therapeutically effective amount of the CDKL5fusion protein may range from about 1pg to about 10 g. In some embodiments, a therapeutically effective amount of a CDKL5fusion protein or a pharmaceutical composition containing such a CDKL5fusion protein can range from about 10nL to about 10 mL.

For some embodiments, a therapeutically effective amount may be from about 20 to about 50ng per injection, such as for an intraventricular injection. In other embodiments, the therapeutically effective amount may be about 10 microliters per injection, such as for an intraventricular injection. In further embodiments, the therapeutically effective amount may be about 5ng/μ L, such as for an intraventricular injection. In yet another embodiment, for intraventricular injection, the therapeutically effective amount may be about 1.9 μ g/kg body weight.

In other embodiments, the therapeutically effective amount may be from about 1 to about 2 micrograms per injection, such as for injections given systemically. In further embodiments, the therapeutically effective amount may be about 200 to about 300 μ Ι _ per injection, such as for injections given systemically. In some embodiments, the therapeutically effective amount may be about 5ng/μ L, such as for a systemic injection. For some embodiments, a therapeutically effective amount may be from about 1 to about 1.5 μ g/5g body weight. In some embodiments, the therapeutically effective amount may be from about 200 μ g to about 300 μ g/kg body weight.

In embodiments where the auxiliary active agent is contained in a pharmaceutical formulation in addition to the CDKL5fusion protein, the therapeutically effective amount of the auxiliary active agent will vary depending on the auxiliary active agent. In some embodiments, the effective amount of the auxiliary active agent ranges from 0.001 micrograms to about 1 milligram. In other embodiments, the effective amount of the auxiliary active agent ranges from about 0.01IU to about 10000 IU. In further embodiments, the effective amount of the auxiliary active agent ranges from 0.001mL to about 1 mL. In still other embodiments, the effective amount of the auxiliary active agent ranges from about 1% w/w to about 50% w/w of the total pharmaceutical formulation. In further embodiments, the effective amount of the auxiliary active agent ranges from about 1% v/v to about 50% v/v of the total pharmaceutical formulation. In still other embodiments, the effective amount of the auxiliary active agent ranges from about 1% w/v to about 50% w/v of the total pharmaceutical formulation.

Dosage forms

In some embodiments, the pharmaceutical formulations described herein can be in dosage form. The dosage form may be adapted for administration by any suitable route. Suitable routes include, but are not limited to, oral (including buccal and sublingual), rectal, epidural, intracranial, intraocular, inhalation, intranasal, topical (including buccal, sublingual, or transdermal), vaginal, intraurethral, parenteral, intracranial, subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, intraosseous, intracardiac, intraarticular, intracavernous (intracavernous), intrathecal, intravitreal, intracerebral and intracerebroventricular, and intradermal. Such formulations may be prepared by any method known in the art.

Dosage forms suitable for oral administration may be discrete dosage units such as capsules, pills or tablets, powders or granules, solutions or suspensions in aqueous or non-aqueous liquids; edible foams or foam bodies (wings), or in the form of oil-in-water liquid emulsions or water-in-oil liquid emulsions. In some embodiments, a pharmaceutical formulation suitable for oral administration further comprises one or more agents that flavor, preserve, color, or aid in dispersing the pharmaceutical formulation. Dosage forms prepared for oral administration may also be in the form of liquid solutions that can be delivered as a foam, spray, or liquid solution. In some embodiments, an oral dosage form may contain about 1ng to 1000g of a pharmaceutical formulation containing a therapeutically effective amount, or an appropriate portion thereof, of a CDKL5fusion protein or a composition containing a CDKL5fusion protein. The oral dosage form can be administered to a subject in need thereof.

Where appropriate, the dosage forms described herein may be microencapsulated. Dosage forms may also be prepared to prolong or sustain the release of any ingredient. In some embodiments, the CDKL5fusion protein is a delayed release component. In other embodiments, the release of the optionally included adjunct ingredient is delayed. Suitable methods for delaying the release of the ingredient include, but are not limited to, coating or embedding the ingredient in the material with a polymer, wax, gel, or the like. Sustained release dosage formulations may be prepared as described in standard references such as: "Pharmaceutical dosage form tablets (Pharmaceutical dosage form tablets)", Leberman (Liberman) et al (New York), Marsel Dekker, Inc. (Marcel Dekker, Inc.), 1989), "Remington-The science and practice of medicine (Remington-The science and practice of medicine)", 20 th edition, Risperket Williams and Wilkins publishing (Lippincott Williams & Wilkins), Baltimore (Baltimore), MD, 2000, and "Pharmaceutical dosage form and drug delivery System (Pharmaceutical dosage form and drug delivery systems)", 6 th edition, Anxiel (Ansel) et al (P.P.A. handbarrow PA (1995) and Williams, 1995). These references provide information on the excipients, materials, equipment and methods used to prepare tablets and capsules, and sustained release dosage forms of tablets and pills, capsules and granules. Sustained release can be anywhere from about one hour to about 3 hours or more.

Examples of suitable coating materials include, but are not limited to, cellulosic polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic polymers and copolymers, and also under the trade nameMethacrylic resins, zeatin, shellac and polysaccharides are commercially available from the Rohm Pharma, Westerstadt, Germany.

Coatings may be formed with varying ratios of water soluble polymers, water insoluble polymers and/or pH dependent polymers in the presence or absence of water insoluble/water soluble non-polymeric excipients to produce the desired release profile. The coating is performed on dosage forms (matrix or simple) including, but not limited to, tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particulate compositions, formulated as, but not limited to, suspensions or as "as is ingredient" of sprinkled dosage forms.

Formulations suitable for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. In some embodiments, the pharmaceutical formulation is applied as a topical ointment or cream for treatment of the eye or other external tissue, such as the mouth or skin. When formulated in an ointment, the CDKL5fusion protein, co-active ingredient, and/or pharmaceutically acceptable salt thereof may be formulated with a paraffin or water-miscible ointment base. In other embodiments, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Dosage forms suitable for topical administration in the mouth include lozenges, pastilles and mouthwashes.

Dosage forms suitable for nasal or inhalation administration include aerosols, solutions, suspended drops, gels or dry powders. In some embodiments, the CDKL5fusion protein, the composition containing the CDKL5fusion protein, the co-active ingredient, and/or a pharmaceutically acceptable salt thereof in a dosage form suitable for inhalation is in a reduced particle size form obtained or obtainable by micronization. In some embodiments, the particle size of the size-reduced (e.g., micronized) compound or salt or solvate thereof is defined by a D50 value of about 0.5 to about 10 microns as measured by suitable methods known in the art. Dosage forms suitable for administration by inhalation also include fine particle dusts or mists. Suitable dosage forms in which the carrier or excipient is a liquid for administration as a nasal spray or drops include aqueous or oily solutions/suspensions of the active ingredient, which may be produced by various types of metered dose pressurised nebulisers, nebulisers or insufflators.

In some embodiments, the dosage form is an aerosol formulation suitable for administration by inhalation. In some of these embodiments, the aerosol formulation contains a solution or fine suspension of the CDKL5fusion protein, a composition containing the CDKL5fusion protein, and/or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable aqueous or non-aqueous solvent. Aerosol formulations can be provided in sealed containers in single or multiple doses in sterile form. For some of these embodiments, the sealed container is a single or multi-dose nasal or aerosol dispenser equipped with a metered dose valve (e.g., a metered dose inhaler) that can be discarded once the contents of the container are depleted.

Where the aerosol dosage form is contained in an aerosol dispenser, the dispenser contains a suitable propellant, such as compressed air, carbon dioxide or an organic propellant (including but not limited to hydrofluorocarbons), under pressure. Other embodiments of the aerosol formulation are contained in a pump nebulizer. Pressurized aerosol formulations may also contain solutions or suspensions of CDKL5fusion proteins, compositions containing CDKL5fusion proteins, or pharmaceutical formulations thereof. In further embodiments, the aerosol formulation further contains co-solvents and/or modifiers incorporated to improve, for example, the stability and/or taste and/or microparticle quality characteristics (amount and/or profile) of the formulation. Administration of the aerosol formulation may be once daily or several times daily, for example 2, 3, 4 or 8 times daily, with 1, 2 or 3 doses delivered per time.

For some dosage forms suitable and/or suitable for administration by inhalation, the pharmaceutical formulation is an inhalable dry powder formulation. Such dosage forms may contain, in addition to the CDKL5fusion protein, the composition containing the CDKL5fusion protein, the auxiliary active ingredients and/or pharmaceutically acceptable salts thereof, a powder base such as lactose, glucose, trehalose, mannitol and/or starch. In some of these embodiments, the CDKL5fusion protein, the composition containing the CDKL5fusion protein, the co-active ingredient, and/or the pharmaceutically acceptable salt thereof are in a reduced particle size form. In further embodiments, the performance modifier is a metal salt such as L-leucine or another amino acid, cellobionate octaacetate, and/or stearic acid, such as magnesium stearate or calcium stearate.

In some embodiments, the aerosol formulation is arranged such that each metered aerosol dose contains a predetermined amount of an active ingredient, such as one or more of the CDKL5fusion proteins or compositions containing CDKL5fusion proteins described herein.

Dosage forms suitable for vaginal administration may be provided as pessaries, tampons, creams, gels, pastes, foams or spray formulations. Dosage forms suitable for rectal administration include suppositories or enemas.

Dosage forms suitable for parenteral administration and/or for any injection type (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, intraosseous, epidural, intracardiac, intraarticular, intracavernosal, intrathecal, intravitreal, intracerebral and intracerebroventricular) can include aqueous and/or non-aqueous sterile injection solutions which can contain antioxidants, buffers, bacteriostats, solutes which render the composition isotonic with the blood of the subject, and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents. Dosage forms suitable for parenteral administration may be provided in single unit dose or multi-unit dose containers, including but not limited to sealed ampoules or vials. These doses can be lyophilized and resuspended in a sterile carrier to reconstitute the dose prior to administration. In some embodiments, extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

Dosage forms suitable for ocular administration may include aqueous and/or non-aqueous sterile solutions, which may optionally be suitable for injection, and which may optionally contain antioxidants, buffers, bacteriostats, solutes that render the composition isotonic with fluids contained in or surrounding the eye of a subject, and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents.

For some embodiments, the dosage form contains a predetermined amount of CDKL5fusion protein or a composition containing CDKL5fusion protein per unit dose. In one embodiment, the predetermined amount of the CDKL5fusion protein or the composition comprising the CDKL5fusion protein is a therapeutically effective amount of the CDKL5fusion protein or the composition comprising the CDKL5fusion protein for treating or preventing a CDKL5 deficiency, Rett syndrome, and/or symptoms thereof. In other embodiments, the predetermined amount of the CDKL5fusion protein or the composition containing the CDKL5fusion protein may be a suitable fraction of a therapeutically effective amount of the active ingredient. Thus, such unit doses may be administered once or more than once daily. Such pharmaceutical formulations may be prepared by any method well known in the art.

Treatment of neurological disorders using TATk-CDKL5 compositions and formulations

The CDKL5fusion proteins and pharmaceutical formulations thereof described herein may be used to treat and/or prevent a disease, disorder, syndrome, or symptom thereof in a subject. In some embodiments, the CDKL5fusion proteins and pharmaceutical formulations thereof may be used to treat and/or prevent CDKL5 deficiency, Rett syndrome, variants of Rett syndrome, and/or symptoms thereof. In some embodiments, the subject has a CDKL5 deficiency, Rett syndrome, a variant of Rett syndrome, and/or a symptom thereof.

An amount of the CDKL5fusion proteins, compositions, and pharmaceutical formulations thereof described herein can be administered to a subject in need thereof one or more times daily, weekly, monthly, or yearly. In some embodiments, the amount administered may be a therapeutically effective amount of the CDKL5fusion protein, composition, and pharmaceutical formulations thereof. For example, the CDKL5fusion proteins, compositions, and pharmaceutical formulations thereof may be administered in daily doses. This amount may be given as a single dose per day. In other embodiments, the daily dose may be administered in multiple doses per day, with each dose containing a fraction of the total daily dose to be administered (sub-dose). In some embodiments, the amount of dose delivered per day is 2, 3, 4, 5, or 6. In further embodiments, the compound, formulation, or salt thereof is administered one or more times per week, such as 1, 2, 3, 4, 5, or 6 times per week. In other embodiments, the CDKL5fusion proteins, compositions, and pharmaceutical formulations thereof may be administered one or more times per month, such as 1 to 5 times per month. In still further embodiments, the CDKL5fusion proteins, compositions, and pharmaceutical formulations thereof may be administered one or more times per year, such as 1 to 11 times per year.

The CDKL5fusion proteins, compositions, and pharmaceutical formulations thereof may be co-administered with the second agent by any convenient route. The second agent is a compound and/or formulation separate from the CDKL5fusion protein, composition, and pharmaceutical formulations thereof. The second agent can be administered concurrently with the CDKL5fusion protein, the composition, and pharmaceutical formulations thereof. The second agent can be administered sequentially with the CDKL5fusion protein, the composition, and pharmaceutical formulations thereof. The second agent can have an additive or synergistic effect with the CDKL5fusion protein, the composition, and pharmaceutical formulations thereof. Suitable second agents include, but are not limited to, DNA, RNA, amino acids, peptides, polypeptides, antibodies, aptamers, ribonucleases, ribonuclease guide sequences, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatory agents, antihistamines, anti-infective agents, and chemotherapeutic agents that inhibit translation and transcription of essential tumor proteins and genes. In some embodiments, the second agent is DCA.

Suitable hormones include, but are not limited to, amino acid derived hormones (e.g., melatonin and thyroxine), small peptide and protein hormones (e.g., thyroid stimulating hormone releasing hormone, antidiuretic hormone, insulin-like, growth hormone, luteinizing hormone, follicle stimulating hormone, and thyroid stimulating hormone), eicosanoids (e.g., arachidic acid, lipoxin, and prostaglandins), and steroid hormones (e.g., estradiol, testosterone, tetrahydrotestosterone cortisol).

Suitable immunomodulators include, but are not limited to, prednisone, azathioprine, 6-MP, cyclosporine, tacrolimus, methotrexate, interleukins (e.g., IL-2, IL-7, and IL-12), cytokines (e.g., interferons (e.g., IFN- α, IFN- β, IFN- κ, IFN- ω, and IFN- γ), granulocyte colony stimulating factor, and imiquimod), chemokines (e.g., CCL3, CCL26, and CXCL7), cytosine phosphate-guanosine, oligodeoxynucleotides, dextran, antibodies, and aptamers).

Suitable antipyretics include, but are not limited to, non-steroidal anti-inflammatory drugs (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), aspirin and related salicylates (e.g., choline salicylate, magnesium salicylate, and sodium salicylate), acetaminophen/acetaminophen, metacamizomib, nabumetone, antipyrine, and quinine.

Suitable anxiolytics include, but are not limited to, benzodiazepines (e.g., alprazolam, bromocriptine, chlordiazepoxide, clonazepam, lorazepam, oxazepam, temazepam, triazolam and tofisopam), 5-hydroxytryptamine antidepressants (e.g., selective 5-hydroxytryptamine reuptake inhibitors, tricyclic antidepressants and monoamine oxidase inhibitors), mepivacaine, afobazole, selank, broumantan, 3-hydroxy-6-methyl-2-ethylpyridine hydrochloride, azaperone, barbiturates, hydroxyzine, pregabalin, valdol, and beta blockers.

Suitable antipsychotic agents include, but are not limited to, benproperidol, bromperidol, haloperidol, moperone, pipamperone, timiperone, fluspirilene, pentafluridol, pimozide, acepromazine, chlorpromazine, cyamemazine, dizyrazine, fluphenazine, levomepromazine, thiamphenizine, perprazine, piperazinine, perphenazine, pipothiazine, prochlorperazine, promazine, promethazine, thioproperdine, methylthiodazine, trifluoperazine, chlorpromazine, chlorprothixene, haloperidol, thiothixene, tetiazol, zulothiol, clothiapine, cleipine, prothioxapride, carpesine, procapramide, molindone, mosaprimide, sulpiride, verapride, amisulpiride, amosulpiride, amoxapine, aripiproline, aripiprazole, aride, meprobine, aripiprazole, meprobine, meprobamate, mep, Nemorubide, olanzapine, paliperidone, perospirone, quetiapine, remopride, risperidone, sertindole, trimipramine, ziprasidone, zotepine, alstonie, percentile, bitopidine, epiprazole, cannabidiol, cariprazine, pimavanserin, pomumetad methionil, pencarilin, xanomeline, and kirilopine.

Suitable analgesics include, but are not limited to, acetaminophen/acetaminophen, non-steroidal anti-inflammatory drugs (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), opioids (e.g., morphine, codeine, oxycodone, hydrocodone, dihydromorphine, meperidine, buprenorphine), tramadol, norepinephrine, flupirtine, nefopam, oxyphennaridimine, pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone, ketonidone, piperazinium, and aspirin, as well as related salicylates (e.g., choline salicylate, magnesium salicylate, and sodium salicylate).

Suitable antispasmodics include, but are not limited to, mebeverine, papaverine, cyclobenzaprine, carisoprodol, oxfenadrin, tizanidine, metaxalone, methocarbamol, chlorzoxazone, baclofen, dantrolene, baclofen, tizanidine, and dantrolene.

Suitable anti-inflammatory agents include, but are not limited to, prednisone, non-steroidal anti-inflammatory drugs (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), and immunoselective anti-inflammatory derivatives (e.g., submandibular peptide-T and derivatives thereof).

Suitable antihistamines include, but are not limited to, H1-receptor antagonists (e.g., acrivastine, azelastine (azelastine), bilastine (bilastine), brompheniramine, buclizine, brompheniramine, carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine, clemastine (clematine), cyproheptadine, desloratadine, dexbrompheniramine (dexbrompheniramine), dexchlorpheniramine, dimenhydrinate, diphenhydramine, diammine (doxylamine), ebastine (ebasine), enbranamine (embramine), fexofenadine, hydroxyzine, levocetirizine (levocetirizine), loratadine (loratadine), meloxicam, milnacipramine, olazine, oxfenfenadine, pheniramine (e), trimetrezine (84), pyrilamine (e), trimethoprim, Famotidine, lafutidine, nizatidine (nizatidine), ranitidine (rafatidine) and roxatidine (roxatidine)), tritoquinoline, catechin (catehin), cromoglycate, nedocromil, and beta 2-adrenergic agonists.

Suitable anti-infective agents include, but are not limited to, anti-amebiases (nitazoxanide, paromomycin, metronidazole, tinidazole, chloroquine, miltefosine, amphotericin b, and diiodoquine), aminoglycosides (e.g., paromomycin, tobramycin, gentamicin, amikacin, kanamycin, and neomycin), anthelmintics (e.g., pyrantel, mebendazole, ivermycin, praziquantel, albendazole, thiabendazole, oxaniquine), antifungal agents (e.g., azole antifungal agents (e.g., itraconazole, fluconazole, posaconazole, ketoconazole, clotrimazole, miconazole, and voriconazole), echinomycin (e.g., caspofungin, anidulafungin, and micafungin), griseofulvin, terbinafine, flucytosine, and polyenes (e.g., nystatin and amphotericin b)), antimalarials (e.g., pyrimidine/sulfadoxine, clomazine, amikacin, and amikacin), artemether/lumefantrine, atovaquone/proguanil, quinine, hydroxychloroquine, mefloquine, chloroquine, doxycycline, pyrimethamine and halofantrine), anti-tuberculosis agents (e.g., aminosalicylates), isoniazid/rifampin, isoniazid/pyrazinamide/rifampin, bedaquiline, isoniazid, ethambutol, rifampin, rifapentine, capreomycin and cycloserine), antiviral agents (e.g., amantadine, abacavir/lamivudine, emtricitabine/tenofovir, bitratavir/emtricitabine/tenofovir, efavir/tenofovir, abacavir/lamivudine/zidovudine, lamivudine/zivudine, emtricitabine/tenofovir, fluvovirgindovudine/tenofovir, Emtricitabine/lopinavir/ritonavir/tenofovir, interferon alpha 2 v/ribavirin, pegylated interferon alpha-2 b, maraviroc, raltegravir, deloglivir, emfovir, foscarnet, fomivirsen, oseltamivir, zanamivir, nevirapine, efavirenz, etravirine, rilpivirine, delavirdine, nevirapine, entecavir, lamivudine, adefovir, sofosbuvir, didanosine, tenofovir, avarvr, zidovudine, stavudine, emtricitabine, zalcitabine, tipivdine, cidivir, bocivir, telaprevir, pirovir/ritonavir, fosamprenavir, ritonavir, telonavir, tipinavir, nelfinavir, indinavir, quinavir, fosalvir, fosalvudine, valacivir, valaciclovir, acyclovir, famciclovir, ganciclovir and valganciclovir), carbapenems (e.g. doripenem, meropenem, ertapenem and cilastatin/imipenem), cephalosporins (e.g. cefadroxil, cephradine, cefazolin, cephalexin, cefepime, ceftaroline, chlorocepham, cefotetan, cefuroxime, cefoxitin, cefaclor, ceftibuten, ceftizoxime, cefpodoxime, cefdinir, cefixime, cefditoren, ceftizoxime and ceftazidime), glycopeptide antibiotics (e.g. vancomycin, dalbavancin, oritin and telavancin), glycinyclines (e.g. tegafur), antileprosy drugs (e.g. clofazimine and saryamine), lincomycin and derivatives thereof (e.g. clindamycin and lincomycin), Macrolides and their derivatives (e.g., telithromycin, fidaxomycin, erythromycin, azithromycin, clarithromycin, dirithromycin and oleandomycin), linezolid, sulfamethoxazole/trimethoprim, rifaximin, chloramphenicol, fosfomycin, metronidazole, aztreonam, bacitracin, penicillins (amoxicillin, ampicillin, carbenicillin, piperacillin, ticarcillin, amoxicillin/clavulanic acid, ampicillin/sulbactam, piperacillin/tazobactam, clavulanic acid/ticarcillin, penicillins, procainamicin, oxacillin, dicloxacillin and nafcillin), quinolones (e.g., lomefloxacin, norfloxacin, ofloxacin, qtofloxacin, moxifloxacin, ciprofloxacin, levofloxacin, gemifloxacin, moxifloxacin, cinoxacin, doxafloxacin, doxorfloxacin, antibiotic, doxorfloxacin, nalidixic acid, enoxacin, grexacin, gatifloxacin, trovafloxacin and sparfloxacin), sulfonamides (e.g. sulfamethoxazole/trimethoprim, sulfasalazine and sulfisoxazole), tetracyclines (e.g. doxycycline, demecycline, minocycline, doxycycline/salicylic acid, doxycycline/omega-3 polyunsaturated fatty acids and tetracycline) and urinary tract anti-infectives (e.g. furadadine, urotropin, fosfomycin, cinoxacin, nalidixic acid, trimethoprim and methylene blue).

Suitable chemical agents include, but are not limited to, paclitaxel, Belntezumab, doxorubicin, 5-FU (fluorouracil), everolimus, pemetrexed, melphalan, pamidronate sodium, anastrozole, exemestane, nelarabine, ofatumumab, bevacizumab, belinostat, tositumomab, carmustine, bleomycin, bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib, bicalutamide, lomustine, daunorubicin, clofarabine, cabozantinib, dactinomycin, ramucirumab, arabinoside, carcinostat, cyclophosphamide, decitabine, dexamethasone, docetaxel, hydroxyurea, amiloride, leuprolide, epirubicin, oxaliplatin, amidase, estramustine, cetuximab, imodulcimide, visfatin, giberellin bacillus, amianthrin, amitsunamide, amifosteride, and the like, Etoposide, flutamide, toremifene, fulvestrant, letrozole, degarelix, pralatrexate, methotrexate, floxuridine, obituzumab, gemcitabine, afatinib, imatinib mesylate, carmustine, eribulin, trastuzumab, altretamine, topotecan, panatinib, idarubicin, ifosfamide, ibrutinib, axitinib, interferon alpha-2 a, gefitinib, romidepsin, ixabepilone, ruxolitinib, cabazitaxel, ado-trastuzumab, enrofloxacin, carfilzomib, chlorambucil, sargracitabine, mitotane, vincristine, procarbazine, megestrol, trimetinib, mesna, strontium-89 chloride, chlorambucil, mitomycin, busulfan, tolytin, vinorelbine, milbexabexat, trexat, trexatin, tretinomycin, bexatin, trexatin, temozastin, Felgrastine glycolated, sorafenib, nilutamide, pentostatin, tamoxifen, mitoxantrone, pemetrexed, dinicodulin-toxin linker, alitretinol, carboplatin, pertuzumab, cisplatin, pomalidomide, prednisone, aldesleukin, mercaptopurine, zoledronic acid, lenalidomide, rituximab, octreotide, dasatinib, regorafenib, histrelin, sunitinib, cetuximab, omastatin, thioguanine (thioguanine), dalanib, erlotinib, bexadine, temozolomide, thiotepa, polyamines, BCG, sirolimus, bendamustine hydrochloride, triptorelin, arsenic trioxide, lapatinib, valrubicin, panitumumab, vinblastine, bortezomib, pavemic acid, azacitidine, teniposide, teicoplanin, folinic acid, tetanib, folin, folinic acid, pteridipine, and folic acid, Capecitabine, enzalutamide, ipilimumab, goserelin, vorinostat, esalaisis, ceritinib, abiraterone, epothilone, tafluoroprost, azathioprine, doxifluridine, vindesine, and all-trans retinoic acid.

In embodiments where the CDKL5fusion protein, composition, and pharmaceutical formulation thereof are co-administered simultaneously with the second agent, the CDKL5fusion protein, composition, and pharmaceutical formulation thereof can be administered to the subject at substantially the same time as the second agent. As used in this context, "substantially the same time" refers to the administration of the CDKL5fusion protein, composition, and pharmaceutical formulation thereof, and the second agent, wherein the time period between the administration of the CDKL5fusion protein, composition, or pharmaceutical formulation thereof, and the second agent is between 0 and 10 minutes.

In embodiments where the CDKL5fusion protein, composition, or pharmaceutical formulation thereof is co-administered with the second agent sequentially, the CDKL5fusion protein, composition, or pharmaceutical formulation thereof may be administered first, and then the second agent is administered after a period of time. In other embodiments in which the CDKL5fusion protein, composition, or pharmaceutical formulation thereof is co-administered with the second agent sequentially, the second agent may be administered first, and then the CDKL5fusion protein, composition, or pharmaceutical formulation thereof is administered after a period of time. In any embodiment, the time period between administration of the CDKL5fusion protein, composition, or pharmaceutical formulation thereof and the second agent may be between from 10 minutes to about 96 hours. In some embodiments, the time period may be about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, or about 12 hours. Sequential administration may be repeated as necessary over the course of a treatment cycle.

The amounts of CDKL5fusion proteins, compositions, pharmaceutical formulations thereof that can be administered are described elsewhere herein. The amount of the second agent will vary depending on the second agent. The amount of the second agent can be a therapeutically effective amount. In some embodiments, the effective amount of the second agent ranges from 0.001 micrograms to about 1 milligram. In other embodiments, the amount of the second agent ranges from about 0.01IU to about 10000 IU. In further embodiments, the amount of the second agent ranges from 0.001mL to about 1 mL. In still other embodiments, the amount of the second agent ranges from about 1% w/w to about 50% w/w of the total pharmaceutical formulation. In further embodiments, the amount of the second agent ranges from about 1% v/v to about 50% v/v of the total pharmaceutical formulation. In still other embodiments, the amount of the second agent ranges from about 1% w/v to about 50% w/v of the total second agent composition or pharmaceutical formulation.

In some embodiments, the composition or formulation containing the CDKL5fusion protein is administered to the patient via injection. Suitable injection methods include, but are not limited to, intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, intraosseous, epidural, intracardiac, intraarticular, intracavernosal, intrathecal, intravitreal, intracerebral and intracerebroventricular injection. Other suitable methods of administration of the compositions or formulations containing the CDKL5fusion protein include, but are not limited to, topical, transdermal, nasal, or oral delivery. In some embodiments, the dose of the CDKL5fusion protein ranges from about 0.01 μ g/g body weight to about 10mg/g body weight.

In other embodiments, the CDKL5fusion protein may be delivered to a patient in need of treatment via cell therapy. Turning to fig. 3, this fig. 3 illustrates one embodiment of a method of delivering a CDKL5fusion protein via autologous cells. The method begins by culturing cells in vitro (8000). Preferably, the cells are autologous cells. In one embodiment, the autologous cells are neurons or neuronal precursor cells, such as neural stem cells. In some embodiments, the autologous cells are neurons derived from induced pluripotent stem cells. In other embodiments, the autologous cells are neurons derived from cord blood stem cells.

Subsequently, the cultured cells were transduced with the purified CDKL5fusion protein (8010). In other embodiments, the cultured cells are transduced by exposing the cultured cells to a medium containing the CDKL5fusion protein as previously described. In further examples, cultured cells are transfected with a suitable vector containing CDKL5fusion protein cDNA. The cells were then cultured for an appropriate amount of time to allow expression of the CDKL5fusion protein (8020). In some embodiments, the cells are cultured for about 6 hours to about 96 hours. Following cell culture, one or more transduced cells are administered to the patient.

In one embodiment, the transduced autologous neurons are delivered to the brain using surgical techniques. In some embodiments, the one or more transduced cells are administered to the patient via injection. In some embodiments, one or more transduced cells are included in the formulation. In one embodiment, the formulation containing the one or more transduced cells further comprises a pharmaceutically acceptable carrier and/or active agent. In some embodiments, the formulation containing the one or more transduced cells is administered to the patient via injection or using surgical techniques.

Kit containing CDKL5fusion protein and preparation thereof

The CDKL5fusion proteins, compositions containing CDKL5fusion proteins, and pharmaceutical formulations thereof described herein may be provided as a combination kit. As used herein, the term "combination kit" or "kit of parts" refers to the CDKL5fusion protein, compositions containing CDKL5fusion protein, and pharmaceutical formulations thereof described herein as well as additional components for packaging, selling, marketing, delivering, and/or administering the combination of elements or single elements contained therein, such as an active ingredient. Such additional components include, but are not limited to, packages, syringes, blister packs, bottles, and the like. When one or more components (e.g., active agents) contained in the kit are administered simultaneously, the combination kit may contain the active agents in a single pharmaceutical formulation (e.g., a tablet) or in separate pharmaceutical formulations.

The combination kit may contain the individual agents, compositions, pharmaceutical formulations or components thereof in separate compositions or pharmaceutical formulations. The separate compositions or pharmaceutical formulations may be contained in a single package or in separate packages within the kit. Buffers, diluents, solubilizing agents, cell culture media, and other reagents are also provided in some embodiments. These additional components may be contained in a single package or in separate packages within the kit.

In some embodiments, the combination kit further comprises instructions printed on or otherwise contained in the tangible expression medium. The instructions may provide information on the content of the CDKL5fusion protein, the composition containing the CDKL5fusion protein and pharmaceutical formulations thereof, and/or other adjuvants and/or secondary agents contained therein, safety information on the content of the CDKL5fusion protein, the composition containing the CDKL5fusion protein and pharmaceutical formulations thereof, and/or other adjuvants and/or secondary agents contained therein, information on the dose, use indication and/or one or more recommended treatment regimens of the CDKL5fusion protein, the composition containing the CDKL5fusion protein, and pharmaceutical formulations thereof, and/or other adjuvants and/or secondary agents contained therein. In some embodiments, the instructions may provide instructions for administering the CDKL5fusion protein, compositions containing the CDKL5fusion protein, and pharmaceutical formulations thereof, and/or other adjuvants and/or second agents to a subject having a CDKL5 deficiency, Rett syndrome, and/or symptoms thereof.

Without further explanation, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent. It is emphasized that the embodiments of the present disclosure, particularly any "preferred" embodiments, are merely possible examples of implementations, merely set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the disclosed embodiment(s) of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are within the scope of the present disclosure.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and the publications cited are hereby incorporated by reference to disclose and describe the methods and/or materials. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method may be performed in the order of events recited or in any other order that is logically possible.

Unless otherwise indicated, embodiments of the present disclosure will employ techniques of molecular biology, microbiology, nanotechnology, organic chemistry, biochemistry, botany, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

Examples of the invention

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed and claimed herein. The following examples are to be considered only as illustrative and not limiting in any way to the remainder of the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is in degrees celsius and pressure is at or near atmospheric pressure. The standard temperature and pressure are defined as 20 ℃ and 1 atmosphere.

Example 1: production and purification of TATk-CDKL5 protein.

To generate a deliverable TAT-CDKL 5fusion protein, a synthetic TAT kappa-PTD was used, in which mutation of the furin recognition sequence in the TAT domain effects secretion of the recombinant protein. Successful uptake of the secreted protein by the target cells was observed. The TATk-CDKL5fusion gene containing human CDKL5 was cloned into the expression plasmid pSecTag2 (Life Technologies). This plasmid is designed to enable expression of the gene in a mammalian host and high expression levels of the target protein. The protein expressed from pSecTag2 was fused at the N-terminus to the murine Ig kappa chain leader sequence for protein secretion in culture. TATk-CDKL5fusion proteins were labeled with GFP proteins to allow western blot analysis using anti-GFP antibodies. To facilitate protein purification, the TATk-CDKL5fusion protein was constructed to include a myc tag and a 6xHis tag at the C-terminal region of the TATk-GFP-CDKL5 gene. HEK293T cells were transfected with the TATk-GFP-CDKL5 expression plasmid using standard plasmid delivery methods. After transfection, cells were grown in serum-free Medium (Dulbecco's Modified Eagle Medium) with high glucose. After 48 hours, the medium was collected, diafiltered and concentrated using Amicon ultracentrifuge filters (50kDa cut-off). This method allows buffer exchange and enrichment of secreted proteins.

Fig. 4A and 4B show results of western blot analysis of TATk-GFP-CDKL5 protein expression from transfected HEK293T cells. Figure 4A shows TATk-GFP-CDKL 5fusion protein expression in cell homogenates from transfected HEK293T cells. FIG. 4B shows TATk-GFP-CDKL 5fusion protein aggregation in concentrated (20X) cell culture media from transfected HEK293T cells.

Example 2: verification of TATk-CDKL5 kinase activity.

To purify the TATk-GFP-CDKL5 protein, a myc tag and a 6XHis tag were added to the C-terminal region of the TATk-GFP-CDKL5 gene. TATk-GFP-CDKL 5fusion protein was purified from the culture medium on Ni-NTA resin. CDKL5 kinase has been shown to have high autophosphorylation activity. As shown in fig. 5A and 5B (which show results from in vitro kinase activity assays), the purified TATk-GFP-CDKL5 protein retained its autophosphorylation activity. This demonstrates that the purified fusion protein retains its kinase activity.

Example 3: internalization of TATk-CDKL5 by HEK293T cells.

To evaluate the transduction efficiency of the TATk-GFP-CDKL 5fusion protein, HEK293T cells were incubated with purified/concentrated fusion protein. Briefly, TATk-GFP-CDKL 5fusion proteins were produced and purified as described in example 1. HEK293 cells were incubated in concentrated medium containing the fusion protein. After different incubation times, cells were lysed and the total protein extract was transferred to nitrocellulose membranes for immunoblotting to quantify the TATk-GFP-CDKL5 protein. As shown in figure 6, TATk-GFP-CDKL5 was internalized by the cells after only about 30 minutes of incubation. Other cultures were treated in parallel and fixed, immunostained with anti-GFP specific antibodies to visualize the transduced TATk-GFP-CDKL5 protein. As shown in fig. 7A-7B, the TATk-GFP-CDKL5 protein was efficiently translocated into the cell. Internalization in target cells was confirmed by confocal microscopy (fig. 8). SH-SY5Y neuroblastoma cells were incubated for 30 minutes in a concentrated medium containing the fusion protein. Fig. 8 shows images of a series of confocal images (1-12) of SH-SY5Y cells transduced with TATk-GFP-CDKL5, demonstrating that the TATk-GFP-CDKL5 protein is internalized by the target cell and is located in both the nucleus and cytoplasm of SH-SY5Y cells (fig. 8).

Example 4: TATk-CDKL5 induces differentiation and inhibits proliferation of SHSY5Y neuroblastoma cell line

Although CDKL5 is of clear importance for the central nervous system, the biological function of this kinase remains largely unknown. The CDKL5 gene affects both proliferation and differentiation of neural cells (see, e.g., Waley (Valli) et al, 2012, Biochemicla and biophysics Acta (Biochim Biophys), 1819:1173-1185, and Rizzi et al, 2011, Brain Studies (Brain Res.), 1415: 23-33). Neuroblastoma cells share several features with normal neurons and are therefore considered to be good in vitro models for studying the biochemical and functional properties of neuronal cells, especially when they are induced to differentiate after treatment with agents such as Retinoic Acid (RA) (see, for example, xingh, 2007 brain studies, 1154, pages 8-21; Melino, 1997, journal of neurooncology (j. neuroool.), 31, pages 65-83). For these reasons, neuroblastoma (neuroblastoma) cells were used to study CDKL5 function in vitro.

SH-SY5Y cells were treated with purified TATk-GFP-CDKL5 similar to the treatment described in example 3. Herein, SH-SY5Y cells were incubated with concentrated medium containing purified TATk-GFP-CDKL5 protein for about 24 hours. Cell proliferation was assessed as the mitotic index (the ratio between the number of populations in cells undergoing mitosis and the number of non-mitogenic populations in cells) using hoechst nuclear staining. Differentiation was assessed by examining neurite outgrowth, which is a marker of neuronal differentiation. For the analysis of neurite outgrowth, cells were grown for an additional 1-2 days with or without the presence of the differentiating agent RA. Neurite outgrowth is measured using an image analysis system.

Induction of CDKL5 expression (via TATk-GFP-CDKL5 protein) resulted in strong inhibition of cell proliferation (e.g., fig. 9A-9B and fig. 10) while not increasing apoptotic cell death compared to controls (data not shown). Furthermore, as shown in fig. 11A-11B and fig. 12, TATk-GFP-CDKL5 promoted neuroblastoma cell differentiation as indicated by neurite outgrowth of SH-SY5Y cells. These results demonstrate that TATk-CDKL5 is functional in an in vitro neuronal model.

Example 5: characterization of CDKL5-KO mouse model

Recently a CDKL5 knockout mouse model (Amindola, 2014, public science library complex (PLoS One), 9(5): e91613) was created by EMBL in Monte-Toton, Italy by a group led by doctor Cornelius Gross (Cornelius Gross). To establish the effect of CDKL 5-loss function on the dendritic development of neonatal neurons, the dendritic morphology of neonatal hippocampal granulosa cells derived from CDKL5KO mice was examined. Immunohistochemical analysis of the dendritic morphology of nascent neurons with double-cortical protein (DCX) takes advantage of the expression of this protein in the cytoplasm of mature neurons during the neurite elongation phase. As shown in fig. 13A-13B, DCX positive cells of CDKL5 knockout mice (-/Y) exhibited a dendritic tree with a highly immature pattern (fig. 13B) compared to the wild-type (+/Y) counterpart (fig. 13A). Highly immature versions can be indicated by few branches and elongations. The absence of CDKL5 resulted in a decrease in the number of DCX-positive cells (fig. 13B) due to increased apoptotic cell death (data not shown), which was observed to affect post-mitotic immature granular neurons (DCX-positive cells) (richchs (Fuchs), 2014, disease neurobiology (Neurobiol Dis.), 70, pages 53-68). This data indicates that CDKL5 has a fundamental role in postnatal neurogenesis by affecting the survival of neural precursors and the maturation of nascent neurons. It was observed that cultures of Neuronal Precursor Cells (NPCs) from the subventricocolar zone (SVZ) of Cdkl5 knockout mice exhibited the same defects observed in vivo in cerebellar granule cell precursors. In other words, in the culture of neuronal precursor cells derived from wild-type mice (+/+), there were more neurons (β -tubulin III positive cells, red cells) than in the culture of neuronal precursor cells derived from CDKL5KO (-/-) mice (fig. 14A and 14B). This indicates that the loss of CDKL5 reduces the survival of post-mitotic neurons. Evaluation of neurite outgrowth in β -tubulin III positive cells demonstrated that the neurons generated by Cdkl5 knockout NPCs were less differentiated compared to wild-type neurons (fig. 14A and 14B). These results indicate that post-mitotic NPCs from CDKL5 knockout mice have intrinsic defects not only in cell survival but also in neuronal maturation.

Example 6: the TATk-CDKL5 protein restored neurite development derived from neuronal cell precursors of CDKL5KO mice.

Neuronal precursor cell cultures from CDKL5KO (-/-) and wild type (+/-) mice were treated with TATk-GFP-CDKL5 or TATk-GFP. By measuring the total neurite length of differentiated neurons (positive for β -tubulin III). The evaluation of neurite length was performed by using image analysis system image Pro Plus (Media Cybernetics, Silver Spring, Maryland (MD), 20910, USA (USA)). The mean neurite length/cell was calculated by dividing the total neurite length by the number of cells counted in the region. As shown in fig. 15A-15C and fig. 16, the absence of CDKL5 decreased maturation of new neurons and treatment with TATk-CDKL5 restored neurite development.

Example 7: delivery of TATk-CDKL5 into mouse brain.

Seven day old mouse pups (single dose corresponding to about 200 μ l of 200x concentrated medium; which may contain about 1-1.5 μ g of fusion protein) were injected subcutaneously with a single dose of medium from HEK293T cells transfected with TATk-GFP-CDKL5, TATk-GFP, or medium from untransfected cells (vehicle). The medium from the transfection was collected after 48 hours and diafiltered and concentrated with an Amicon ultracentrifuge filter (50kDa cut-off). 4 hours after the treatment, the mice were sacrificed. The brain was stored in fixative for 24 hours, cut along the midline and stored in 20% sucrose in phosphate buffer for an additional 24 hours. The hemispheres were frozen and stored at-80 ℃. The right hemisphere was cut into a 30 μm thick coronal section with a cryomicrotome. Immunohistochemistry was performed on free-floating sections. The localization of TATk-GFP-CDKL5 and TATk-GFP in brain was assessed by immunohistochemistry using anti-GFP antibody and TSA amplification kits. Images were acquired at the level of the sensory-motor cortex and cerebellum. Cells were counterstained with 4', 6-diamidino-2-phenylindole (DAPI). Representative images showing the presence of the TATk-GFP-CDKL5 protein in the sensory-motor cortex and cerebellum of mice are shown in figures 17A-17F and figures 18A-18D, respectively. Given that the TATk-GFP-CDKL5 protein was administered subcutaneously, this data demonstrates that the TATk-GFP-CDKL5 protein is efficiently transported across the blood brain barrier and into brain cells.

Example 8: effect of TATk-CDKL5fusion protein on neuronal maturation, survival and connectivity in vivo

Intracerebroventricular injections of adult mice (4-6 months of age) were performed with either TATk-GFP-CDKL5 or TATk-GFP for 5 consecutive days (fig. 19) (see, e.g., the experimental schedule of fig. 20). Briefly, mice were anesthetized with chloraminone (100-125 mg/kg) and xylazine (10-12.5 mg/kg). Cannulae (0.31mm diameter, brain perfusion kit III; Alzet, Cupertino, Calif. (CA)) were implanted stereotactically into the lateral ventricles (A/P-0.4 mm tail, M/L1.0mm, D/V-2.0 mm; FIG. 19). Seven days after implantation, mice were perfused with 10 μ l (about 50ng) of TATk-GFP-CDKL5 or TATk-GFP in PBS for 5 consecutive days by using a Hamilton syringe connected to a motorized nanoinjector (at a rate of 0.5 μ l/min). Four hours after the final injection, animals were sacrificed and the dendritic morphology of the neonatal hippocampal granulocytes was analyzed by immunohistochemistry using DCX. FIGS. 21 and 22 demonstrate that DCX-positive neurons from Cdkl5KO mice have shorter processes (FIGS. 21A-21B and 22A-22B) compared to DCX-positive neurons from wild-type counterparts. It was observed that intraventricular administration of TATk-GFP-CDKL 5fusion protein for five consecutive days increased the neurite length and number of neurite branches in CDKL5 knockout mice (fig. 22C) to levels similar to wild type (fig. 22A). FIGS. 23A-23B show examples of reconstituted dendritic trees of novacells of: wild type (+/Y) (fig. 23A), hemizygous CDKL5 knockout mice (-/Y) (fig. 23B) and hemizygous CDKL5 knockout male mice treated with TATk-GFP-CDKL 5fusion protein.

Quantification of the dendritic size of DCX positive cells demonstrated that CDKL5 knockout mice (-/Y) had shorter dendritic length (fig. 24A) and reduced number of segments (fig. 24B) compared to wild type mice (fig. 24A and 24B). In TATk-GFP-CDKL5 treated CDKL5 knockout mice (—/Y), there was an increase in both parameters, even greater compared to +/Y mice (fig. 24A-24B). The effect of TATk-GFP-CDKL5 treatment on the details of the dendrite architecture was examined by evaluating each dendrite grade separately. A significant feature of CDKL5KO mice was the absence of higher branches (fig. 25A-25B; red arrows). While wild-type (+/Y) mice had branches up to grade 10, CDKL5 knockout mice (—/Y) lacked branches at grade 8-10 (fig. 25A, arrows). In addition, the CDKL5 knockout mouse (-/Y) showed decreased branch length of 5-8 stages (FIG. 25A) and decreased branch number of 6-8 stages (FIG. 25B). Taken together, these data indicate that in Cdkl5KO mice, the dendritic trees of neonatal granulocytes have developmental disorders and this defect is due to a reduction in the number and length of intermediate branches and the absence of higher branches. All these defects were observed to be completely rescued by TATk-GFP-CDKL5 treatment (fig. 25A to 25B).

To evaluate the effect of TATk-GFP-CDKL5 treatment on apoptotic cell death, we counted the number of apoptotic cells expressing cleaved caspase-3 in the hippocampal dentate gyrus (fig. 26). Quantification of cleaved caspase-3 cells showed that TATk-GFP-CDKL5 treatment completely normalized apoptotic cell death in CDKL5 knockout mice (/ Y) (fig. 26). CDKL5 knockout mice (-Y) were observed to have fewer post-mitotic neurons (DCX positive cells) in the hippocampal dentate gyrus than wild-type (+/Y) mice (fig. 27). TATk-GFP-CDKL5 treated CDKL5 knockout mice experienced an increase in the number of post-mitotic neurons and thereby became similar to the number of post-mitotic neurons in wild-type (+/Y) mice (fig. 27). This indicates that increased death of post-mitotic immature granulosa cells characterizing CDKL5 knockout mice was rescued by TATk-GFP-CDKL5 treatment. Taken together, these data demonstrate that treatment with TATk-GFP-CDKL5 in CDKL5 knockout mice increases neurite length and survival of novacells in hippocampus, suggesting that injected TATk-CDKL5 spreads from the lateral ventricle to hippocampus and restores maturation and survival of granulosa cells post-mitotic.

Without being bound by any one theory, the decrease in connectivity may be corresponding to a dendritic developmental disorder characterizing the neonatal granulosa cells of CDKL5KO mice. Synaptophysin (SYN; also known as p38) is a synaptophysin that is a specific marker for presynaptic terminals. Here, a significantly lower optical density of SYN in the hippocampus molecular layer was observed in CDKL5 knockout mice (/ Y) than in wild-type (+/Y) mice (fig. 28 and 30A), indicating that there was less synaptic connectivity in the dentate gyrus of CDKL5KO mice. Fig. 28A-28C show representative images of brain sections treated for Synaptophysin (SYN) immunofluorescence displaying a layer of Dentate Gyrus (DG) molecules (Mol) from: wild type male mice (+/Y) (fig. 28A), hemizygous CDKL5 knockout male mice (-/Y) (fig. 28B), and hemizygous CDKL5 knockout male mice treated with TATk-GFP-CDKL 5fusion protein via intraventricular injection given once daily for 5 consecutive days (-/Y + TATk-GFP-CDKL5) (fig. 28C). One sixth of the 30 μm thick coronal sections of DG from animals were processed for immunohistochemistry. Immunohistochemistry was performed on free-floating sections of frozen brains. For synaptophysin immunohistochemistry, sections were incubated with mouse monoclonal anti-SYN (SY38) antibody (1:1000, MAB 5258, Millipore Bioscience research reagent) for 48 hours at 4 ℃ and with Cy3 conjugated anti-mouse IgG secondary antibody (1: 200; Jackson Immunoresearch) for 2 hours. The intensity of Immunoreactivity (IR) was determined by densitometry of immunohistochemically stained sections. Fluorescence images were captured using a Nikon Eclipse E600 microscope equipped with a Nikon (Nikon) digital camera DXM1200(ATI system). Densitometric analysis of the molecular and cortical layers was performed using Nis-element software 3.21.03 (nikon). For each image, the intensity threshold is estimated by analyzing the distribution of pixel intensities that do not contain IR in the image region. This value is then subtracted to calculate the IR for each sampled area. This value is given as a percentage of the optical density of the control CDKL5+/Y mice (mean + standard error).

Dendritic branching of cortical pyramidal neurons was significantly reduced in CDKL5 knockout mice compared to wild-type counterparts (almondo, 2014, public science library complex, 9(5): e 91613). A similar lower level of SYN immunoreactivity was observed in layer III of neocortex (fig. 30B). In CDKL5 knockout mice treated with TATk-GFP-CDKL5(—/Y), these defects were completely rescued (fig. 28 and 30A and 30B), indicating that treatment with TATk-GFP-CDKL5 had a positive effect on dendritic structure concurrent with restoration of input to neurons.

Example 9: effect of TATk-CDKL5fusion protein on P-AKT in vivo

AKT is a central signaling kinase associated with multiple cellular pathways. Phosphorylated AKT (P-AKT) was significantly reduced in CDKL5 knockout animals, CDKL5 deficiency and Rett syndrome. Fig. 29A-29C show representative images of brain slices displaying treatment of the Dentate Gyrus (DG) molecular layer (Mol) from: wild type male mice (+/Y) (fig. 29A), CDKL5 knockout male mice (-/Y) (fig. 29B), and CDKL5 knockout male mice treated with TATk-GFP-CDKL 5fusion protein via intraventricular injection given once daily for 5 consecutive days (-/Y + TATk-GFP-CDKL5) (fig. 29C). For phospho-AKT immunohistochemistry, sections were incubated with the mouse monoclonal anti-phospho-AKT-Ser 473 antibody (1:1000, Cell Signaling Technology) for 24 hours at 4 ℃ and with a Cy 3-conjugated anti-mouse IgG secondary antibody (1: 200; Jackson Immunol research) for 2 hours. The intensity of Immunoreactivity (IR) was determined by densitometry of immunohistochemically stained sections. Fluorescence pictures were captured using a Nikon Eclipse E600 microscope equipped with a Nikon (Nikon) digital camera DXM1200(ATI system).

It was observed that the optical density of P-AKT in the DG molecular layer (fig. 31A) and in the cortical V layer (fig. 31B) was significantly lower in CDKL5 knockout male mice (—/Y) than in +/Y mice. This defect was completely rescued in CDKL5 knockout male mice (—/Y) injected intracerebroventricularly for five consecutive days with TATk-GFP-CDKL5 (fig. 31A and 31B), demonstrating that TATk-GFP-CDKL5 treatment in CDKL5 knockout mice restored AKT activity.

Example 10: influence of TATk-CDKL5fusion protein on learning ability and memory ability

CDKL5 knockout mice exhibit learning and memory deficits compared to wild-type mice (see, e.g., fig. 33 and fig. 34A-34B).

To examine memory and learning, CDKL5 knockout mice were given daily intracerebroventricular injections of TATk-GFP-CDKL 5fusion protein for 10 consecutive days (see, e.g., the experimental schedule of fig. 32). After a two-day rest period at the end of the 10-day injection, mice in all groups received the Morris Water Maze (MWM) test (fig. 33). MWM measures the ability to find and recall the position of a platform immersed in water. In the MWM task, mice were trained to find hidden escape platforms in a round pool of water. The device consists of a large circular water tank (1.00m diameter, 50cm height) with a transparent circular escape platform (10cm 2). The pool was divided into essentially four equal quadrants, which were identified as northeast, northwest, southeast and southwest. The water tank was filled with tap water at a temperature of 22 ℃ up to 0.5cm above the top of the platform and the water was made opaque using milk. The platform was placed in the tank in a fixed position (in the middle of the northwest quadrant). The sink was placed in a large room with many internal (square, triangular, circular and star) and external maze visual cues. After training, each mouse was tested for two phases, 4 times per day, each for 5 consecutive days with 40 min intervals between phases (acquisition phase). The video camera was placed over the center of the pool and connected to a video tracking system (Ethovision 3.1; nodassian information Technology B.V.), Wageningen (Wageningen), the Netherlands (Netherlands). Mice were released towards the pool wall starting from the following starting points: north, east, south or west and allow mice to find the platform for up to 60 seconds. If the mouse does not find the platform, it is gradually guided to the platform and left there for 15 seconds. During the test interval (inter-trail time) (15 seconds), mice were placed in empty cages. The latency of finding the hidden platform is taken as a measure of learning. All experimental phases were performed between 9.00 am and 15.00 pm.

The results of this test are shown in fig. 33. Figure 33 shows a graph demonstrating the quantification of the learning period as determined via the morris water maze test for the following: wild type male mice (+/Y), CDKL5 knockout male mice (-/Y), and CDKL5 knockout male mice treated with TATk-GFP-CDKL 5fusion protein (-/Y + TATk-GFP-CDKL 5). Wild type mice learned the search platform by day two, but no significant learning was detected in CDKL5 knockout mice. CDKL5 knockout mice treated with TATk-CDKL5fusion protein restored learning capacity beginning on day four and continued to improve on day 5.

The memory and learning ability in response to TATk-GFP-CDKL 5fusion protein treatment was further examined using a passive avoidance test. After 10 consecutive days of treatment and two days of rest, different groups of mice received passive avoidance testing (fig. 34). The experiment utilized a test cage with two chambers (light and dark). On the first day (conditioning period), animals were placed in the light chamber and instinctively moved into the dark chamber where they suffered a single adverse event (foot shock). For the passive avoidance test, we used a tilted floor box (47 × 18 × 26cm) divided into two compartments by a sliding door and a control unit incorporating a shock absorber (Ugo Basile, Italy). This classical instrument for pavlov conditioning exploits the tendency of mice to escape from illuminated areas to dark areas (evasive experimental approach). On the first day, mice were placed into the lighting compartments separately. After a 60 second acclimation period, the connecting door between the chambers was opened. Generally, mice quickly step through the gate and into the dark compartment, as mice prefer to be in the dark. After entering the dark compartment, the mice received a brief foot shock (0.7mA for 3 seconds) and were removed from the chamber after a latency time of 15 seconds. If the mice linger in the photopic compartment for the duration of the test (358s), the gate is closed and the mice are removed from the photopic compartment. Between tests of individual mice, the chamber was cleaned with 70% ethanol. After a 24 hour retention period, the mice were placed back into the photopic compartment and the measured time it took them to re-enter the dark compartment (latency) was up to 358 seconds.

FIGS. 34A-34B show results from passive avoidance testing. Fig. 34A shows that the latency into the dark chamber is similar for all groups. On the next day (test period) (fig. 34B), animals were again placed in the light chamber. Memory for adverse events was determined by the latency of entering the dark chamber. The CDKL5 knockout mouse (-/Y) was severely impaired in completing this task as evidenced by the reduced latency into the dark compartment compared to wild type mice (+/Y). TATk-GFP-CDKL5 treated knockout mice exhibited similar latency compared to wild type mice.

Taken together, the data demonstrate that TATk-CDKL5 can increase learning and memory in CDKL5 knockout mice and restore to similar levels seen in untreated wild-type mice.

Example 11: influence of TATk-CDKL5fusion protein on motor function

CDKL5 knockout mice exhibit prolonged limb clasping when suspended (see, e.g., fig. 35A-35B).

To examine the effect of TATk-GFP-CDKL 5fusion protein on motor function, mice were given daily intracerebroventricular injections of TATk-GFP-CDKL5 for 10 consecutive days (fig. 35). 10 days after completion of the dosing regimen, mice were suspended in the air by tail (fig. 35A and 35B). All mice were suspended for about 2 minutes and the total time for limb closure was measured. The results from this experiment are shown in FIGS. 35A-35B.

Figure 35A shows a graph demonstrating quantification of locomotor ability as determined by the hugging test, where the total amount of time taken for limbs to hug during a 2 minute interval was measured: wild type male mice (+/Y), CDKL5 knockout male mice (-/Y), and CDKL5 knockout male mice treated with TATk-GFP-CDKL 5fusion protein according to the injection schedule in figure 32(-/Y + TATk-GFP-CDKL 5).

Body weights of wild-type male mice (+/Y) and CDKL5KO (-/Y) male mice injected with TAT-GFP-CDKL5 protein for 5(+/Y) or 10(-/Y) days were measured and the results are shown in fig. 36. No significant changes in body weight were observed during the injection period, indicating the absence of any side effects caused by administration of TAT-GFP-CDKL5 protein.

In conclusion, the data demonstrate that treatment with TATk-CDKL5 improves motor function in CDKL5 knockout mice.

Claims (20)

1. A fusion protein comprising:

a CDKL5 polypeptide sequence, wherein the CDKL5 polypeptide sequence has about 50% to 100% sequence identity to SEQ ID NO 2 or SEQ ID NO 16; and

a TATk polypeptide sequence, wherein the TATk polypeptide sequence has about 90% to about 100% sequence identity to SEQ ID No. 4, wherein the TATk polypeptide is operably linked to the CDKL5 polypeptide.

2. The fusion protein of claim 1, further comprising an Igk-chain leader sequence polypeptide, wherein the Igk-chain leader sequence is operably linked to the CDKL5 polypeptide.

3. The fusion protein of claim 1, further comprising a reporter protein polypeptide, wherein the reporter protein polypeptide is operably linked to the CDKL5 polypeptide.

4. The fusion protein of claim 1, further comprising a protein tag polypeptide, wherein the protein tag polypeptide is operably linked to the CDKL5 polypeptide.

5. The fusion protein of claim 1, wherein the fusion protein has a polypeptide sequence according to SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12 or SEQ ID No. 14.

6. The fusion protein of claim 1, wherein the fusion protein increases neurite growth, neurite elongation, neurite branch number, or neurite branch density in the brain of a subject compared to a control.

7. The fusion protein of claim 1, wherein the fusion protein reduces neuronal apoptosis in the brain of the subject compared to a control.

8. A pharmaceutical formulation comprising:

a therapeutically effective amount of a fusion protein comprising:

a CDKL5 polypeptide sequence, wherein the CDKL5 polypeptide sequence has about 50% to 100% sequence identity to SEQ ID NO 2 or SEQ ID NO 16; and

a TATk polypeptide sequence, wherein the TATk polypeptide sequence has about 90% to about 100% sequence identity to SEQ ID No. 4, wherein the TATk polypeptide is operably linked to the CDKL5 polypeptide; and

a pharmaceutically acceptable carrier.

9. The pharmaceutical formulation of claim 8, wherein the fusion protein further comprises an Igk-chain leader sequence polypeptide, wherein the Igk-chain leader sequence is operably coupled to the CDKL5 polypeptide.

10. The pharmaceutical formulation of claim 8, wherein the fusion protein further comprises a reporter protein polypeptide, wherein the reporter protein polypeptide is operably coupled to the CDKL5 polypeptide.

11. The pharmaceutical formulation of claim 8, wherein the fusion protein further comprises a protein tag polypeptide, wherein the protein tag polypeptide is operably coupled to the CDKL5 polypeptide.

12. The pharmaceutical formulation of claim 8, wherein the fusion protein has a polypeptide sequence according to SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, or SEQ ID NO 14.

13. The pharmaceutical formulation of claim 8, wherein the therapeutically effective amount of the fusion protein treats one or more symptoms of a CDKL5 deficiency, Rett syndrome, or Rett syndrome variant in a subject as compared to a control.

14. The pharmaceutical formulation of claim 8, wherein the therapeutically effective amount of the fusion protein increases neurite growth, neurite elongation, number of neurite branches, or neurite branch density in the brain of a subject compared to a control.

15. The pharmaceutical formulation of claim 8, wherein the therapeutically effective amount of the fusion protein reduces neuronal apoptosis in the brain of the subject compared to a control.

16. The pharmaceutical formulation of claim 8, wherein the therapeutically effective amount of the fusion protein improves motor function in a subject compared to a control.

17. The pharmaceutical formulation of claim 8, wherein the therapeutically effective amount of the fusion protein improves cognitive function in a subject compared to a control.

18. A method, comprising:

administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical formulation comprising:

an amount of a fusion protein comprising:

a CDKL5 polypeptide sequence, wherein the CDKL5 polypeptide sequence has about 50% to 100% sequence identity to SEQ ID NO 2 or SEQ ID NO 16; and

a TATk polypeptide sequence, wherein the TATk polypeptide sequence has about 90% to about 100% sequence identity to SEQ ID No. 4, wherein the TATk polypeptide is operably linked to the CDKL5 polypeptide; and

a pharmaceutically acceptable carrier.

19. The method of claim 18, wherein the subject in need thereof has or is suspected of having a CDKL5 deficiency, Rett syndrome, or a Rett syndrome variant.

20. The method of claim 18, wherein the therapeutically effective amount of the fusion protein treats one or more symptoms of a CDKL5 deficiency, Rett syndrome, or Rett syndrome variant in a subject as compared to a control.