Treatment for CDKL5 deficiency

Abstract

Disclosed herein are methods and agents for the treatment and / or prevention of cyclin-dependent kinase-like 5 (CDKL5) deficiency. In particular, disclosed herein are compounds or compositions for increasing cyclin-dependent kinase-like 2 (CDKL2) in a subject, for example, in the brain of a subject, and the use of such compounds and compositions in methods for treating CDKL5 deficiency.

Description

Technical Field

[0001] The present invention relates to the treatment and / or prevention of CDKL5 deficiency by a compound or composition that increases the expression or activity of cyclin-dependent kinase-like 2 (CDKL2).

Background Art

[0002] Brain development is an incredibly complex and intricate process, and any disruption within it can lead to tragic consequences. When such problems occur, the resulting conditions are labeled as neurodevelopmental disorders (NDDs) and may prevent reaching cognitive, emotional, and motor development milestones. As a result, a patient's motor function, learning, communication, etc. may be permanently impaired [1]. To date, they have been shown to affect more than 3% of the world's children, and there are few available effective treatments [2].

[0003] Clearly, these disorders are a true concern in our society, and it should be a priority to understand them better. However, finding treatments for these diseases has been difficult for various reasons, such as delayed diagnosis, disease complexity and heterogeneity, or the lack of animal models. Despite these challenges, some successful treatments have been developed thanks to the continuous efforts of researchers and clinicians. One of the most important recent achievements is the gene therapy Zolgensma® which has revolutionized the treatment for spinal muscular atrophy, a rare and fatal genetic disease that affects the survival of motor neurons [3]. Other treatments for spinal muscular atrophy include antisense oligonucleotides, nusinersen (Spinraza®), and the small molecule therapy, risdiplam (Evrysdi).

[0004] Other diseases in that group are expected to progress in a similar manner. One of these conditions that has not yet been successfully addressed is CDKL5 deficiency (CDD), which is characterized by early-onset seizures and severe neurodevelopmental disorders and is caused by mutations in a serine / threonine kinase called cyclin-dependent kinase-like 5 (CDKL5).

[0005] In contrast to more common non-disorders (NDDs) such as autism spectrum disorder (ASD), which was first described in 1911, CDD is a relatively recent discovery.[4] It wasn't until 2003 that CDKL5 mutations observed in two girls were associated with infantile seizures and intellectual disability.[5] Prior to that, CDD was classified as either an early-onset seizure variant or a Hanefeld variant of Rett syndrome.[6]

[0006] Despite increasing attention to CDKL5 and growing evidence linking CDKL5 mutations to symptoms, CDD was not considered a distinct clinical entity until 2013. This was simply due to the discovery of a distinct clinical profile for CDD when comparing the clinical features of patients with CDD and patients with Rett syndrome.[7]

[0007] In 2018, the first Phase 3 clinical trial, including patients with CDD, began, testing the efficacy of an antiepileptic compound called ganaxolone. In 2022, ganaxolone became the only FDA-approved treatment specifically indicated for CDD. CDD was finally classified by the World Health Organization in 2020, leading to greater visibility of the disorder in clinical research and enabling patients' caregivers to obtain insurance coverage.

[0008] The prevalence of CDD is currently estimated at approximately 1 in 40,000–60,000 births, but the situation regarding CDD remains unchanged recently, and new cases are still being identified at a rapid pace [8, 9, 10]. CDD patients are predominantly female, with male patients accounting for only 1:4 of the affected population. Furthermore, male patients tend to be more severely affected, suggesting that CDKL5 mutations are primarily fatal in developing males [11, 12, 10].

[0009] NDD is often caused by a combination of various environmental factors (i.e., infection, malnutrition, social deprivation, or physical trauma) and genetic factors. CDD, however, is one of the rare NDDs caused solely by a gene mutation. CDD is the result of a de novo mutation in the CDKL5 gene located on the short arm of the X chromosome (Xp22). One familial case of CDKL5 mutation has been reported in two siblings, which was likely due to maternal germline mosaicism

[13] .

[0010] More than 265 pathogenic mutations have been reported on CDKL5, half of which were point mutations. 38 percent of these point mutations are missense mutations, representing the most common mutations in CDD patients

[14] . Furthermore, all of these missense mutations (with one exception) are located on the catalytic domain of CDKL5, and patients exhibit more severe symptoms. This increased severity demonstrates the importance of CDKL5 activity in the disease [15, 16, 10]. In addition, 13 percent of CDD patients have frameshift mutations due to point insertions or deletions [17, 12]. Several cases of partial or complete deletions of CDKL5 have also been reported, and even a case of partial deletion on the short arm of the X chromosome has been identified [18, 19, 20, 21].

[0011] CDKL5 duplication has also been reported in several patients, all of whom exhibited autistic features, developmental delay, language impairment, and hyperactivity, but lacked epilepsy, a major clinical manifestation of CDD

[22] . These cases suggest that CDKL5 may be dose-sensitive and that misregulation of CDKL5 expression has significant effects on brain development. Finally, two genetically identical twin siblings were diagnosed with CDD in 2015 with the same mutation, but their phenotypes were very different

[23] , which could be explained by X chromosome inactivation. However, this last case could also be explained by environmental or epigenetic factors, opening up the possibility of a broader explanation of CDD beyond purely genetic causes. [Overview of the project]

[0012] The present invention provides compounds or compositions for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2).

[0013] In particular, the compounds or compositions described herein are suitable for increasing the expression or activity of CDKL2 in the brain.

[0014] In some embodiments, the compound or composition comprises a nucleic acid molecule, such as a small interfering RNA (siRNA), guide RNA (gRNA), or short hairpin RNA (shRNA). In some embodiments, the compound or composition comprises a nucleic acid molecule encoding the CDKL2 polypeptide. In some embodiments, the nucleic acid molecule encoding CDKL2 encodes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the polypeptide of SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encoding CDKL2 encodes a polypeptide comprising the sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encoding CDKL2 encodes a polypeptide consisting of the sequence of SEQ ID NO: 1.

[0015] In some embodiments, the nucleic acid molecule encoding CDKL2 comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with a nucleotide sequence selected from the list consisting of SEQ ID NO: 2 and SEQ ID NO: 3. In some embodiments, the nucleic acid molecule encoding CDKL2 comprises

[0016] In some embodiments, the compound or composition includes a viral vector. In some embodiments, the viral vector includes one or more adenovirus vectors, adeno-associated virus vectors, lentiviral vectors, or retroviral vectors. In some embodiments, the nucleic acid molecule further includes a promoter.

[0017] In some embodiments, the compound or composition comprises one or more clustered, regularly arranged short palindromic repeat (CRISPR)-related nucleases, transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZNFs). In some embodiments, the compound or composition comprises a CRISPR-related protein (Cas protein) and a guide RNA (gRNA) that hybridizes within the CDKL2 genomic sequence.

[0018] In some embodiments, the CDKL2 genome sequence is defined by the Ensembl reference ENSG00000138769 (SEQ ID NO: 8).

[0019] In some embodiments, the compound or composition comprises an isolated CDKL2 polypeptide. In some embodiments, the compound or composition comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% relative to the polypeptide of SEQ ID NO: 1. In some embodiments, the compound or composition comprises the polypeptide of SEQ ID NO: 1. In some embodiments, the compound or composition consists of the polypeptide of SEQ ID NO: 1.

[0020] In some embodiments, the compound or composition comprises or further comprises a small molecule activator for CDKL2 expression or activity. In some embodiments, the compound or composition comprises a small molecule activator that specifically binds to a protein structure as defined in PDB database reference 4BBM. In some embodiments, the compound or composition comprises or further comprises a polypeptide that increases CDKL2 expression. In some embodiments, the compound or composition comprises a transcription factor or enhancer that increases CDKL2 expression. In some embodiments, the compound or composition comprises or further comprises an antibody. In some embodiments, the compound or composition comprises a fusion protein comprising a CDKL2 polypeptide operatively linked to a second polypeptide sequence. In some embodiments, the second polypeptide sequence comprises a nuclear localization signal (NLS), a nuclear export signal (NES), a leader signal polypeptide, or a cell-permeable polypeptide.

[0021] In some embodiments, the compound or composition increases CDKL2 activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% compared to CDKL2 activity measured in the absence of the compound or composition. In some embodiments, CDKL2 activity is measured by the level of phosphorylation of the CDKL2 substrate and / or the level of CDKL2 autophosphorylation.

[0022] In some embodiments, CDKL2 activity is measured by the level of phosphorylation of microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2), microtubule-associated protein 1S (MAP1S), or voltage-gated R-type calcium channel subunit α-1E (CACNA1E). In some embodiments, microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2) contains or consists of the amino acid sequence of SEQ ID NO: 5, microtubule-associated protein 1S (MAP1S) contains or consists of the amino acid sequence of SEQ ID NO: 6, or voltage-gated R-type calcium channel subunit α-1E (CACNA1E) contains or consists of the amino acid sequence of SEQ ID NO: 7.

[0023] In some embodiments, CDKL2 activity is measured by the level of phosphorylation of microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2), which optionally contains or consists of the amino acid sequence of SEQ ID NO: 5.

[0024] In some embodiments, the compound or composition increases CDKL2 expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% compared to CDKL2 expression measured in the absence of the compound or composition. In some embodiments, CDKL2 expression is measured by Western blotting, quantitative real-time PCR (qRT-PCR), advanced RNA sequencing, microarrays, mass spectrometry, ELISA, or Phos-tag gel electrophoresis.

[0025] In some embodiments, the compound or composition further comprises a pharmaceutically acceptable carrier or diluent. In some embodiments, the compound or composition further comprises a second therapeutic agent useful for treating CDKL5 deficiency. In some embodiments, the second therapeutic agent is selected from the list consisting of clobazam, topiramate, prednisolone / prednisone, adrenocorticotropic hormone, and ganaxolone.

[0026] The present invention also provides a method for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) in subjects requiring such increase, the method comprising administering a compound or composition according to the present invention to the subject. The present invention also provides a method for treating or preventing a disease or disorder in a subject, the method comprising administering a compound or composition according to the present invention to the subject. The present invention also provides a method for treating or preventing a neurological disease or disorder in a subject, the method comprising administering a compound or composition according to the present invention to the subject. The present invention also provides a method for treating or preventing cyclin-dependent kinase-like 5 (CDKL5) deficiency in a subject, the method comprising administering a compound or composition according to the present invention to the subject.

[0027] The present invention also provides a compound or composition according to the present invention for use in a method of doing so in a subject that requires an increase in the expression or activity of cyclin-dependent kinase-like 2 (CDKL2). The present invention also provides a compound or composition described herein for use in a method of increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) in the brain of a subject. The present invention also provides a compound or composition according to the present invention for use in a method of treating or preventing a disease or disorder in a subject. The present invention also provides a compound or composition according to the present invention for use in a method of treating or preventing a neurological disease or disorder in a subject. The present invention also provides a compound or composition according to the present invention for use in a method of treating or preventing cyclin-dependent kinase-like 5 (CDKL5) deficiency in a subject. The present invention also provides a compound or composition described herein for use in a method of treating or preventing cyclin-dependent kinase-like 5 (CDKL5) deficiency in a subject, and the compound or composition is capable of increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) in the brain of the subject.

[0028] The present invention also provides the use of a compound or composition according to the present invention for doing so in a subject that requires an increase in the expression or activity of cyclin-dependent kinase-like 2 (CDKL2). The present invention also provides the use of a compound or composition according to the present invention for treating or preventing a disease or disorder in a subject. The present invention also provides the use of a compound or composition according to the present invention for treating or preventing a neurological disease or disorder in a subject. The present invention also provides the use of a compound or composition according to the present invention for treating or preventing cyclin-dependent kinase-like 5 (CDKL5) deficiency in a subject.

[0029] In some embodiments, the compound or composition is administered intrathecally, intravenously, intracystically, intraventricularly, or parenchymally. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the compound or composition increases the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) in the subject.

[0030] This application also provides a compound or composition according to the present invention for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) in a subject.

[0031] This application also provides a composition comprising a cyclin-dependent kinase-like 2 (CDKL2) activator. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or excipient.

Brief Description of the Drawings

[0032] Next, embodiments of the present invention will be described by way of example only, with reference to the accompanying drawings.

[0033] [Figure 1] CDKL5 activity is present in excitatory and inhibitory neurons. (A) Schematic of different Cre lines used to delete CDKL5 in specific brain cell types. (B) Residual pS222 EB2 was observed in CDKL5 KO (-Y) male mice compared to wild-type littermates (+ / Y), indicating that one or more different kinases can phosphorylate EB2 at Ser222 in addition to CDKL5. [Figure 2]Phosphorylation of several microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2) remains in CDKL5 KO cells. (A) and (B) show the reduction in CDKL5 levels (A) and pSer222 EB2 phosphorylation (B) when CDKL5 is knocked out systemically (KO) or with cell-type-specific drivers. See Figure 1A for the color coding of Cre drivers. The data show that CDKL5 and its activity (pEB2) are present in excitatory (green) and inhibitory (yellow and orange) neurons. CDKL5 activity was not observed in astrocytes (purple). [Figure 3] Phospho-Ser222 is present at low levels in CDKL5 knockout. The residual pS222 EB2 levels in the P8 cortex in systemic CDKL5 knockouts indicate that one or more other kinases can phosphorylate EB2. The absence of pS222 signaling in the EB2 phospho mutant mouse cortex confirms that no background is observed with the pS222 antibody used. [Figure 4] The phospho-Ser222 in rat primary cortical neurons is increased upon okadaic acid (OA) treatment and decreased upon N-methyl-d-aspartate (NMDA) treatment. These data demonstrate that 1 hour of phosphatase inhibitor treatment (okadaic acid / OA) in rat primary neurons induces an increase in pS222 EB2, indicating that this site is dephosphorylated by PP1 / PP2A phosphatases under normal conditions. [Figure 5] Phospho-Ser222 can be increased with okadaic acid (OA) and decreased with N-methyl-d-aspartate (NMDA) in cultured neurons derived from CDKL5 KO mice. In primary cortical cultures prepared from CDKL5 KO embryos, OA treatment increased phosphor-S222, indicating that kinases other than CDKL5 can phosphorylate this site. NMDA treatment reduced the pS222 signal, indicating that NMDA downregulates this phosphorylation and that the signal is not background. (B) is a quantification of the blot shown in (A). [Figure 6]Phospho-Ser222 can be increased with okadaic acid (OA) and decreased with N-methyl-d-aspartate (NMDA) in cultured neurons derived from CDKL5 KO mice. Primary cultures from male wild-type littermate control embryos are shown compared to CDKL5 KO cultures. Parallel display of wild-type and CDKL5 knockout littermate cultures from the cortex shows that pS222 EB2 is much lower in CDKL5 KO, but residual pS222 can still be upregulated by OA and downregulated by NMDA, indicating that some pS222 EB2 may persist and be regulated in CDKL5 KO. (B) is a quantification of the blot shown in (A). (C) shows the presence of a consistent increase in pSer222 EB2 with OA in each culture. [Figure 7] Phosphorylated microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2) of ICK and CDKL2 in HEK293 cells. These data represent a screening test to see if other kinases can phosphorylate pS222 EB2. Kinases were expressed in HEK293 cells. Only CDKL5, ICK, and CDKL2 can phosphorylate EB2. (A) shows a tree of kinases that may potentially have substrates overlapping with CDKL5. (B) shows the ability of different recombinant kinases to phosphorylate EB2. (C) shows the pS222 EB2:total EB2 ratio for each kinase, showing that significant phosphorylation is observed only in CDKL5, ICK, and CDKL2. [Figure 8] In HEK293 cells, ICK and CDKL2 phosphorylate microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2). (A) Western blot and (B) quantification of wild-type and kinase-dead (KD) expression of ICK, CDKL2, and CDKL5 in HEK293 cells (ICK left panel, CDKL2 center panel, CDKL5 right panel). These data demonstrate that all three kinases can phosphorylate S222 on EB2. [Figure 9]CDKL5 KO primary cortical neurons transfected with FLAG-tagged CDKL2, ICK, or CDKL5 show increased phospho-Ser222 in the transfected neurons. These data indicate the expression of kinases CDKL5, ICK, and CDKL2 in primary cortical neurons. pS222 EB2 is specifically increased in neurons expressing these kinases, indicating that CDKL2, ICK, and CDKL5 can phosphorylate EB2 in neurons. [Figure 10] CDKL5 knockout primary neurons infected with CDKL2 shRNA exhibited reduced levels of phosphomicrotubule-associated protein RP / EB family member 2 (MAPRE2 / EB2) after treatment with okadaic acid. CDKL2 was knocked down using primary neurons derived from the cortex of CDKL5 knockout mice by lentiviral expression of shRNAs targeting CDKL2. Both shRNAs induced a reduction in the OA-dependent increase of pEB2 / EB2. The data suggest that CDKL2 is another kinase involved in EB2 phosphorylation in CDKL5 knockout neurons. [Figure 11] ICK expression patterns differ from those of CDKL5, but CDKL2 expression overlaps with CDKL5 expression. Using the official Allen Brain Institute database, single-cell mRNA expression of the target kinases was analyzed in adult human brain cells. ICK and CDKL4 expression are very low in human neurons. Therefore, CDKL2 is the most likely candidate for phosphorylating CDKL5 substrates such as EB2 in neurons. [Figure 12] Exemplary CDKL2 amino acid and RNA sequences. This figure discloses exemplary amino acid and mRNA sequences encoding CDKL2. Sequence ID 1 represents the amino acid sequence of a known isoform of CDKL2 (Uniprot reference: Q92772). Sequence IDs 2 and 3 represent exemplary mRNA sequences encoding CDKL2 (RefSeq references NM_003948 and NM_001330724). [Figure 13]Sequence alignment of human CDKL and RCK kinases. The protein sequences of eight kinases were aligned using ICM software. Red squares highlight amino acids common to CDKL5, ICK, and CDKL2. [Figure 14] Three-dimensional structural alignment of human CDKL and RCK kinases. A. Superposition of three-dimensional protein structures of the kinase domains of CDKL5 (orange), ICK (purple), and CDKL2 (pink) using ICM software. Gatekeeper residues are shown as sticks. B. Two-dimensional protein structure alignment of the kinase domains of human CDKL and RCK kinases using ICM software. [Figure 15] Exemplary sequences of microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2) derived from UniProt reference Q15555-1 (SEQ ID NO: 5), microtubule-associated protein 1S (MAP1S) derived from UniProt reference Q66K74-1 (SEQ ID NO: 6), and voltage-gated R-type calcium channel subunit α-1E (CACNA1E) derived from UniProt reference Q15878-1 (SEQ ID NO: 7). [Figure 16] Description of the cleavage site in genomic DNA, where exon 4 of CDKL2 is excised to generate CDKL2 knockout mice. Exon 4 contains the essential coding sequence for the ATP-binding pocket of CDKL2; without it, the functional kinase cannot be formed. [Figure 17]Western blot analysis of knockout mouse brain lysates. All mice were CDKL5 knockout (KO). CDKL2 was either wild-type (wt), heterozygous knockout (+ / -), or homozygous knockout (- / -). A. Two different ages were analyzed at postnatal day 10 (P10) and P16. EB2 phosphoSer222 levels were used as a measure of CDKL5 signaling. pS222 EB2 was reduced in + / - mice compared to wts, and further reduced in - / - at P10. At P16, comparing WT and - / -, there was a robust reduction in pS222 EB2 in - / - mice. B. PhosphoSer222 EB2 / total EB2 was reduced in CDKL2 homozygous (hom) deletion compared to wild-type (wt) mice. CDKL2 deletion reduces the remaining EB2 phosphorylation in CDKL5 KO to approximately 15% at P10. The upper and lower bands represent EB2 splice isoforms. The upper band of the graph in panel B was quantified. [Figure 18] Compared to wild-type mice, CDKL5 / 2 double knockout mice exhibit reduced spontaneous alternation in the Y-maze. Male and female wild-type mice (n=3 males, n=3 females), CDKL5 knockout mice (n=3 males, n=4 females), and CDKL5 / 2 double knockout mice (n=3 males, n=3 females) were compared in Y-maze performance. One-way ANOVA revealed a significant effect of genotype on Y-maze performance [F(2,16)=5.661, p=0.0138] with a moderate effect size (R²) of 0.4144. Tukey's multiple comparison study demonstrated a significantly reduced rate of alternation in CDKL5 / 2 double knockout mice compared to wild-type mice [p=0.0119]. Significant differences (p<0.05) are indicated by *. Error bars represent standard deviation. [Modes for carrying out the invention]

[0034] To facilitate understanding of the present invention, certain terms are first defined below. Further definitions of these terms and other terms are provided throughout this specification.

[0035] As used herein, the terms “cyclin-dependent kinase-like 2,” “cyclin-dependent kinase-like 2,” or “CDKL2” refer to the amino acid sequence encoded by the CDKL2 gene (HGNC reference 1782). This gene is also known as KKIAMRE or P56. This gene is encoded by the genomic sequence identified by Ensembl reference ENSG00000138769. The transcripts ENST00000307465.9 and ENST00000429927.6 can also be used to define the CDKL2 sequence. In some embodiments, CDKL2 may comprise the amino acid sequence of SEQ ID NO: 1 (Uniprot reference: Q92772). In some embodiments, CDKL2 may consist of the amino acid sequence of SEQ ID NO: 1 (Uniprot reference: Q92772). In some embodiments, CDKL2 may be encoded by an mRNA sequence comprising a sequence selected from either SEQ ID NO: 2 or SEQ ID NO: 3 (RefSeq references NM_003948 and NM_001330724). In some embodiments, the CDKL2 protein may have a structure defined in PDB database reference 4BBM

[24] . In preferred embodiments, CDKL2 comprises the amino acid sequence of SEQ ID NO: 1 (Uniprot reference Q92772).

[0036] As used herein, “compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2)” or “cyclin-dependent kinase-like 2 (CDKL2) activator” refers to any agent that increases the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) polypeptide or protein. In particular, the compound or composition is intended to increase CDKL2 expression or activity in the brain. The increase in expression or activity can be measured by any standard means known in the art, including measuring the increase in the level of polynucleotides encoding CDKL2 (such as CDKL2 mRNA) and / or CDKL2 polypeptide or protein. Methods for quantifying the amount of one or more CDKL2 polynucleotides include fluorescence in situ hybridization (FISH), spectroscopy, quantitative sequencing, quantitative PCR (qPCR), RT-PCR, ddPCR, Southern blotting, or Northern blotting. Methods for quantifying the amount of one or more CDKL2 polypeptide or protein include immunohistochemistry (IHC), spectroscopy, colorimetric detection, Western blotting, mass spectrometry, or ELISA. The level of CDKL2 activation or increased activity induced by a drug that increases CDKL2 expression or activity may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% of CDKL2 expression or activity when measured in the absence of the drug that increases CDKL2 expression or activity. Ideally, the levels of CDKL2 activity or expression should be compared within the same model system (e.g., cell line or tissue) in the presence and absence of the drug that increases CDKL2 expression or activity.

[0037] In some embodiments, increased expression or activity of cyclin-dependent kinase-like 2 (CDKL2) polypeptide or protein can be measured by an increase in the phosphorylation level of the CDKL2 substrate. In some embodiments, the CDKL2 substrate is microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2), microtubule-associated protein 1S (MAP1S), or voltage-gated R-type calcium channel subunit α-1E (CACNA1E). In some embodiments, the CDKL2 substrate is microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2). In some embodiments, the sequence of microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2) is the sequence shown in UniProt reference Q15555-1 (disclosed herein as SEQ ID NO: 5). In some embodiments, the sequence of microtubule-associated protein 1S (MAP1S) is the sequence shown in UniProt reference Q66K74-1 (disclosed herein as SEQ ID NO: 6). In some embodiments, the sequence of voltage-gated R-type calcium channel subunit α-1E (CACNA1E) is the sequence shown in UniProt reference Q15878-1 (disclosed herein as SEQ ID NO: 7). The level of increase in CDKL2 substrate phosphorylation brought about by agents that increase CDKL2 expression or activity may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% of CDKL2 substrate phosphorylation when measured in the absence of the agent that increases CDKL2 expression or activity. Ideally, the levels of CDKL2 substrate phosphorylation should be compared within the same model system (e.g., cell line or tissue) in the presence and absence of the agent that increases CDKL2 expression or activity.In some embodiments, the level of microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2) phosphorylation in the presence of a drug that increases CDKL2 expression or activity may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% of CDKL2 substrate phosphorylation when measured in the absence of the drug that increases CDKL2 expression or activity. Ideally, the levels of CDKL2 substrate phosphorylation should be compared within the same model system (e.g., cell line or tissue) in the presence and absence of a drug that increases CDKL2 expression or activity.

[0038] As used herein, the terms “CDKL5 deficiency” or “CDD” refer to the clinical manifestations of a phenotype associated with a deficiency in CDKL5 expression or activity. In some embodiments, CDD expression may be defined by parameters of the prior art, e.g., those shown in

[10] . In some embodiments, CDD expression may be defined by parameters defined in the prior art, e.g., those defined by the Online Mendelian Inheritance in Man (OMIM) database reference 300672

[25] . In some embodiments, CDD expression may be defined by parameters defined in the prior art, e.g., those defined by the International Classification of Diseases (ICD) database (10th edition) reference G40.42

[26] .

[0039] As used herein, the terms “approximately” or “about” refer to values ​​similar to the stated reference values ​​when applied to one or more target values. In some embodiments, unless otherwise specified or evident from the context, the terms “approximately” or “about” refer to a range of values ​​that fall within or less than 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% in either direction (greater than or less than) the stated reference values ​​(except where such numbers exceed 100% of the possible values).

[0040] As used herein, the term “prevention” means delaying or preventing the onset, development, or progression of a disease, disorder, or condition for a period ranging from a few minutes to an indefinite period. “Prevention” also includes reducing the risk of developing a disease, disorder, or condition. “Prevention” includes, but is not required, the complete avoidance of a disease condition.

[0041] As used herein, the term “treatment” (and also “to treat” or “to treat”) refers to any administration of a therapeutic agent in accordance with a treatment regimen that achieves a desired effect in that it partially or completely alleviates, improves, reduces, decreases the severity of, and / or reduces the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. In some embodiments, the administration of a therapeutic agent in accordance with a treatment regimen correlates with the achievement of the desired effect. Such treatment may be for subjects who do not show signs of the disease, disorder, and / or condition in question, and / or for subjects who show only initial signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be for subjects who show one or more established signs of the disease, disorder, and / or condition in question. In some embodiments, treatment may be for subjects who have been diagnosed with the disease, disorder, and / or condition in question. In some embodiments, treatment may be for subjects who are known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the disease, disorder, and / or condition in question. In preferred embodiments of the present invention, the disease, disorder, and / or condition is CDKL5 deficiency.

[0042] As used herein, the term “equivalent” means a system, set of conditions, effect, or result that is sufficiently similar to a test system, set of conditions, effect, or result to enable a scientifically valid comparison. A person skilled in the art will recognize and understand which system, set of conditions, effect, or result is sufficiently similar to any particular test system, set of conditions, effect, or result described herein to be “equivalent.”

[0043] The term “correlated” as used herein has its usual meaning of “showing correlation with.” Those skilled in the art will understand that two features, items, or values ​​are correlated with each other if they tend to appear and / or change together. In some embodiments, the correlation is statistically significant when its p-value is less than 0.05, and in some embodiments, the correlation is statistically significant when its p-value is less than 0.01. In some embodiments, the correlation is assessed by regression analysis. In some embodiments, the correlation is the correlation coefficient.

[0044] As used herein, the terms “improve,” “increase,” or “decrease,” or their grammatical synonyms, refer to values ​​relative to reference (e.g., baseline) measurements, such as measurements obtained under comparable conditions (e.g., in the same individual before the initiation of the treatment described herein) or measurements in a control individual (or control individuals) in the absence of the treatment described herein.

[0045] As used herein, “polypeptide” is a sequence of at least two amino acids linked to each other by peptide bonds. In some embodiments, a polypeptide may contain at least three to five amino acids, each of which is linked to another amino acid by at least one peptide bond. Those skilled in the art will understand that a polypeptide may optionally contain “unnatural” amino acids or other entities that can still be incorporated into the polypeptide chain.

[0046] As used herein, the term “protein” refers to a molecule comprising a polypeptide (i.e., a sequence of at least two amino acids linked to one another by peptide bonds). Proteins may also contain non-amino acid sites (e.g., glycoproteins, proteoglycans, etc.) and / or may be otherwise processed or modified. Those skilled in the art will understand that a “protein” may be a complete polypeptide chain (with or without a signal sequence) as produced by a cell, or a characteristic portion thereof. Those skilled in the art will understand that a protein may comprise two or more polypeptide chains linked, for example, by one or more disulfide bonds, or associated by other means. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of the various amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, and methylation. In some embodiments, proteins may comprise native amino acids, non-native amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having a length of less than approximately 100 amino acids, less than approximately 50 amino acids, less than 20 amino acids, or less than 10 amino acids.

[0047] As used herein, the terms “subject,” “individual,” or “patient” refer to any organism in which embodiments of the present invention may be used or administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans, insects, parasites, etc.). In preferred embodiments of the present invention, the subject is human.

[0048] As used herein, the terms “target cells” or “target tissue” refer to any cell, tissue, or organism. In preferred embodiments, the target cells or target tissue are liver cells, such as hepatocytes, or liver tissue.

[0049] As used herein, the term “therapeutic regimen” means any method used to partially or completely alleviate, improve, reduce, inhibit, prevent, delay the onset, reduce the severity, and / or reduce the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. This may include, at the discretion of, the administration of one or more doses separated by regular or varying time intervals. In some embodiments, a therapeutic regimen is designed so that its implementation achieves a particular effect, e.g., reduction or elimination of an adverse condition or disease, and / or correlates with the achievement of such a particular effect (e.g., across a population of relevant cells, tissues, or organisms). In some embodiments, the treatment includes administering one or more therapeutic agents simultaneously, sequentially, or at different times, at the same time or over different times. In some embodiments, a “therapeutic regimen” includes genetic methods such as gene therapy, gene disruption, or other methods known to induce or reduce expression (e.g., transcription, processing, and / or translation of a particular gene product, such as a primary transcript or mRNA).

[0050] As used herein, the term “therapeutic dose” refers to the amount of a therapeutic agent that gives a therapeutic effect to a subject being treated with a reasonable benefit-to-risk ratio applicable to any medical treatment. Such a therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject shows signs of or feels an effect). In some embodiments, “therapeutic dose” refers to the amount of a therapeutic agent or composition that is effective in treating, improving, or preventing (e.g., delaying onset) an associated disease or condition, by improving symptoms associated with the disease, preventing or delaying the onset of the disease, and / or reducing the severity or frequency of symptoms of the disease, and / or exhibiting a detectable therapeutic or preventive effect. The therapeutic dose is usually administered in a dosing regimen that may contain multiple unit doses. For any particular therapeutic agent, the therapeutic dose (and / or an appropriate unit dose in an effective dosing regimen) may vary depending, for example, on the route of administration or in combination with other therapeutic agents. Alternatively or additionally, a specific therapeutically effective dose (and / or unit dose) for any particular patient may depend on a variety of factors, including the activity of the particular therapeutic agent used; the specific composition used; the patient's age, weight, general health, sex, and diet; the timing of administration, route of administration, and / or excretion or metabolic rate of the particular therapeutic agent used; the duration of treatment; and similar factors well known in the medical field.

[0051] As used herein, “expression” means the process by which a gene ultimately produces a protein. Expression includes, but is not limited to, transcription, post-transcriptional modifications (e.g., splicing, polyadenylation, 5' cap addition), and translation.

[0052] As used herein, “transcription” or “transcribed” refers to the first step in several steps of DNA-based gene expression, in which a target sequence of DNA is copied to RNA (particularly mRNA) by the enzyme RNA polymerase. During transcription, the DNA sequence is read by RNA polymerase, which produces a complementary antiparallel RNA sequence called the primary transcript.

[0053] As used herein, “target sequence” means the sequence in which a compound is intended to hybridize to produce the desired activity for CDKL2 expression. Oligonucleotides are sufficiently complementary to their target sequences to enable hybridization under physiological conditions.

[0054] As used herein, “nucleic acid base complementarity” or “complementarity” means, with respect to nucleic acid bases, a nucleic acid base that can base-pair with another nucleic acid base. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In both DNA and RNA, guanine (G) is complementary to cytosine (C). In some embodiments, complementary nucleic acid bases mean nucleic acid bases of an oligomeric compound that can base-pair with nucleic acid bases of a target sequence. For example, if a nucleic acid base at a particular position in an oligomeric compound can hydrogen-bond with a nucleic acid base at a particular position in a target sequence, the positions of the hydrogen bonds between the oligomeric compound and the target sequence are considered complementary in their nucleic acid base pairing. Nucleic acid bases with certain modifications can still be nucleic acid base complementarity because they can maintain their ability to pair with the corresponding nucleic acid bases.

[0055] As used herein, “non-complementary” with respect to nucleic acid bases means a pair of nucleic acid bases that do not form hydrogen bonds with each other. As used herein, “complementary” with respect to oligomeric compounds (e.g., linked nucleosides, oligonucleotides) means the ability of such oligomeric compounds or regions thereof to hybridize to a target sequence or a region of the oligomeric compound itself through nucleic acid base complementarity.

[0056] As used herein, “fully complementary” with respect to a nucleic acid sequence, amino acid sequence, or region thereof means that each nucleic acid base of the oligomer compound or region thereof can pair with a nucleic acid base of a complementary nucleic acid target sequence or a self-complementary region of the oligomer compound. Therefore, a fully complementary oligomer compound or region thereof does not contain mismatched or non-hybridized nucleic acid bases with respect to its target sequence or the self-complementary region of the oligomer compound.

[0057] As used herein, “complementarity percentage” means the percentage of nucleic acid bases or amino acids in a nucleic acid sequence or amino acid sequence that are complementary to an isolength portion of the target nucleic acid sequence or amino acid sequence. The complementarity percentage is calculated by dividing the number of nucleic acid bases or amino acids in a nucleic acid sequence or amino acid sequence that are complementary to the nucleic acid base or amino acid at the corresponding position in the target nucleic acid sequence or amino acid sequence by the total length of the nucleic acid sequence or amino acid sequence.

[0058] As used herein, “identity percentage” means the number of nucleic acid bases or amino acids in the first nucleic acid sequence or amino acid sequence that are of the same type (unrelated to chemical modification) as the nucleic acid bases or amino acids at the corresponding positions in the second nucleic acid sequence or amino acid sequence, divided by the total number of nucleic acid bases or amino acids in the first nucleic acid sequence or amino acid sequence.

[0059] As used herein, “modulation” means a change in the quantity or quality of a molecule, function, or activity compared to the quantity or quality of the molecule, function, or activity before modification. For example, modification includes any change that increases (stimulates or induces) or decreases (inhibits or reduces) gene expression.

[0060] As used herein, “region” or “regions” or “portion” or “portions” means a plurality of linked nucleic acid bases or amino acids having the functions or properties defined herein, particularly with respect to the claims and definitions provided herein. Typically, such a region or portion comprises at least 10, at least 11, at least 12, or at least 13 linked nucleic acid bases or amino acids. For example, such a region may contain 13 to 20 linked nucleic acid bases or amino acids, e.g., 13 to 16 or 18 to 20 linked nucleic acid bases or amino acids.

[0061] As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administration to animals. In some embodiments, the pharmaceutically acceptable carrier or diluent is sterile saline. In some embodiments, such sterile saline is pharmaceutical-grade saline.

[0062] As used herein, “amino” includes primary, secondary, and tertiary amino groups. As used herein, “halo” and “halogen” mean atoms selected from fluorine, chlorine, bromine, and iodine.

[0063] The term “comprising” is used herein to mean including the specified method steps or elements, but not to include an exclusive list, and therefore additional steps or elements may exist.

[0064] Furthermore, insofar as the term “includes” is used in either the detailed description or the claims, such term is intended to be inclusive in the same manner as the term “comprising” is used as a transitional term in the claims.

[0065] Clinical definition of CDD The main clinical features of CDD were defined in 2019 to aid in diagnosis. CDD is defined by a pathogenic or highly pathogenic variant of the CDKL5 gene, delayed motor and cognitive development, and epilepsy that begins in the first year of life.

[10]

[0066] Epilepsy is the most prominent clinical feature of CDD, with 90% of patients experiencing epileptic episodes by 3 months of age. These episodes occur daily in 8% of cases and weekly in 20% of cases. Notably, 43.6% of CDD patients experience a honeymoon period between 2.5 months and 6 years in which epilepsy ceases. CDD patients experience three stages of epilepsy throughout their lives: (1) childhood seizures, (2) epileptic encephalopathy, and (3) refractory epilepsy. Childhood seizures are the most common stage of epilepsy in CDD patients, occurring in 23% of patients (stage 1), and occur at some point in life in 81% of patients.

[0067] Another important clinical symptom of CDD, now considered a nearly universal feature, is hypotonia. CDD patients also commonly exhibit cortical visual impairment in 75% of cases, with insufficient eye contact and lack of visual tracking. Furthermore, CDD patients tend to reach important milestones at a slow pace or not at all, resulting in significant developmental delays and intellectual disabilities. For example, only 49% of female patients can walk independently by age 5, and only 7.5% of all female patients can form complete sentences.

[0068] In addition to the main characteristics, some of the affected population also exhibit symptoms that are considered to be indirect consequences of the disease. The most prominent are autistic features, hand stereotyping, gastrointestinal symptoms, orthopedic complications, respiratory and cardiac abnormalities, or sleep disturbances. Gastrointestinal symptoms such as constipation, reflux, and aerophagia are the most common concomitant symptoms, occurring in 86.5% of individuals. Hand stereotyping is another common symptom affecting 80% of CDD patients. 68.5% of patients have scoliosis by age 10, often requiring orthopedic corsets or surgical treatment due to hypotonia and rehabilitation. Caregivers also report sleep disturbances and increased nocturnal awakenings in 63% of CDD patients. Finally, a significant portion of CDD patients have been reported to suffer from respiratory abnormalities and cardiac arrhythmias.

[0069] Similar to independent walking, some variability has been reported in the severity of symptoms in CDD patients, with 23% of patients being able to walk independently and 77% not. Another example is the fact that 33.5% of patients can communicate using words or body language, while the rest are unable to express themselves. This variability may be explained by the localization or type of mutation on CDKL5, but the correlation appears to be limited. However, patients with mutations in or upstream of the catalytic domain appear to demonstrate increased symptom severity [15, 27]. In addition, 8.8% of CDD patients have somatic mosaicism

[28] , and the proportion of mosaicism could explain the observed variability.

[0070] However, the potential link between mosaicism and symptom variability has not yet been established. X chromosome inactivation in female patients may be another reason for variability. Approximately half of their cells express non-functional CDKL5, and the severity of symptoms may vary depending on the cell type expressing the mutant allele. As with mosaicism, a correlation has not yet been found.

[0071] Pharmaceutical composition of drugs As used herein, “pharmaceutical composition” means a mixture of substances suitable for administration to an individual. For example, a pharmaceutical composition may include one or more active agents and a sterile aqueous solution.

[0072] As used herein, “pharmaceutically acceptable salt” means a physiologically and pharmaceutically acceptable salt of a compound, i.e., a salt that retains the desired biological activity of the parent compound and does not impart any undesirable toxicological effects thereto.

[0073] Other aspects of the present invention also relate to compounds or compositions for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) according to the present invention, or cells according to the present invention, and, where appropriate, suitable excipients and additives such as physiological saline, stabilizers, or protease inhibitors, as pharmaceuticals or diagnostic aids.

[0074] kit Any pharmaceutical composition described herein may be provided in a kit. In some cases, the kit includes (a) a container containing the pharmaceutical composition described herein, and optionally (b) informational material. The informational material may be, for example, descriptive, instructional, marketing, or other material relating to the use of the methods and / or agents described herein for therapeutic benefit.

[0075] The informational materials of the kit are not limited to their form. In some cases, the informational materials may include information on the production of a compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) according to the present invention, such as concentration, expiration date, batch or production site information. In other cases, the informational materials may relate to methods for administering a compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) according to the present invention, for example, in a preferred amount, method, or mode of administration (e.g., the dose, dosage form, or mode of administration described herein).

[0076] In some cases, informational materials, such as instructions, may be provided in printed form, such as printed text, drawings, and / or photographs, such as labels or printed sheets. Informational materials may also be provided in other formats, such as Braille, computer-readable materials, video recordings, or audio recordings. In other cases, the informational materials in a kit may include contact information, such as a physical address, email address, website, or telephone number, which the user of the kit can use to obtain substantial information regarding the therapeutic agents and / or their use in the methods described herein. Informational materials may also be provided in any combination of formats.

[0077] In addition to the pharmaceutical composition of the present invention, the kit may contain other components such as solvents or buffers, stabilizers, or preservatives. The kit may also contain further agents, e.g., second or third agents, e.g., other therapeutic agents or other therapeutic compounds or compositions. Components may be provided in any form, e.g., liquid, dry, or lyophilized. Components may be substantially pure (they may be combined together or delivered separately) and / or sterile. If components are provided in a liquid solution, the liquid solution may be an aqueous solution, such as a sterile aqueous solution. If components are provided in a dry form, reconstitution is generally carried out by adding a suitable solvent. Solvents, e.g., sterile water or buffer solutions, may be provided in the kit at the option of their own.

[0078] A kit may include one or more containers for a pharmaceutical composition or other drug. In some cases, a kit may include separate containers, dividers, or compartments for the therapeutic agent and informational material. For example, the therapeutic agent may be contained in a bottle, vial, or syringe, and the informational material may be contained in a plastic sleeve or packet. In other situations, the separate elements of a kit are contained within a single, undivided container. For example, a compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) according to the present invention may be contained in a bottle, vial, or syringe with informational material attached in the form of a label. In some cases, a kit may include a plurality (e.g., packs) of individual containers, each containing one or more unit dosage forms (e.g., dosage forms described herein) of the compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) according to the present invention. A container may contain a unit dose, for example, a unit containing the therapeutic agent. For example, a kit may include a plurality of syringes, ampoules, foil packets, blister packs, or medical devices, each containing a unit dose. The kit container may be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or lightly airtight.

[0079] The kit may optionally include a device suitable for administering a compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) according to the present invention, such as a syringe or other suitable delivery device. The device may be provided pre-filled with a unit dose of the compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) according to the present invention, or it may be empty but suitable for filling.

[0080] vector The present invention also provides vectors comprising one or more nucleic acid molecules disclosed herein, such as vectors encoding CDKL2 DNA or mRNA coding sequences. In some embodiments, the present invention provides a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1. A vector according to the present invention may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1. The vector may be a viral or non-viral vector capable of transporting nucleic acid molecules. In some embodiments, the vector is a plasmid or cosmid (e.g., a circular double-stranded DNA that can ligate additional DNA segments). In some embodiments, the vector is a viral vector that can ligate additional DNA segments into a viral genome. Examples of expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, yeast artificial chromosomes (YACs), Epstein-Barr (EBV)-derived episomes, and other expression vectors known in the art.

[0081] The vectors described herein may include adeno-associated virus (AAV) particles capable of crossing the blood-brain barrier. Such AAVs may also be capable of transducing cells of the central nervous system (CNS), such as neurons. AAVs for this purpose may include mutant or variant capsid proteins known in the art that confer the ability to cross the blood-brain barrier. Compounds or compositions that increase CDKL2 described herein may be delivered as part of a payload in combination with AAV, particularly if the compound or composition is a nucleic acid molecule. Thus, described herein are recombinant adeno-associated virus (AAV) particles comprising an AAV capsid protein and a payload, the payload comprising a compound or composition for increasing the expression or activity of CDKL2, and the recombinant AAV capable of crossing the blood-brain barrier.

[0082] This specification describes recombinant adeno-associated virus (AAV) particles comprising an AAV capsid protein and a nucleic acid payload, wherein the nucleic acid payload encodes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the polypeptide of SEQ ID NO: 1, and the recombinant AAV is capable of crossing the blood-brain barrier.

[0083] This specification describes recombinant adeno-associated virus (AAV) particles comprising an AAV capsid protein and a nucleic acid payload, wherein the nucleic acid payload encodes the polypeptide of Sequence ID No. 1, and the recombinant AAV is capable of crossing the blood-brain barrier.

[0084] This specification describes recombinant adeno-associated virus (AAV) particles comprising an AAV capsid protein and a nucleic acid payload, wherein the nucleic acid payload comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with a nucleotide sequence selected from the list consisting of SEQ ID NOs: 2 and SEQ ID NOs: 3, and the recombinant AAV is capable of crossing the blood-brain barrier.

[0085] This specification describes recombinant adeno-associated virus (AAV) particles comprising an AAV capsid protein and a nucleic acid payload, wherein the nucleic acid payload comprises a nucleotide sequence selected from the list consisting of SEQ ID NOs: 2 and SEQ ID NOs: 3, and the recombinant AAV is capable of crossing the blood-brain barrier.

[0086] CRISPR As used herein, “CRISPR nuclease system” refers collectively to transcripts and other elements involved in directing the expression or activity of CRISPR-related ("Cas") genes, including the Cas gene encoding sequence, guide sequence (also referred to as “spacer” in the context of endogenous CRISPR systems), or other sequences and transcripts from the CRISPR locus.

[0087] In some embodiments, one or more elements of the CRISPR system are derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of the CRISPR system are derived from a specific organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes. The CRISPR system is characterized by elements that facilitate the formation of the CRISPR complex at a site of a target sequence.

[0088] In the context of CRISPR complex formation, the “target sequence” refers to a sequence designed to be complementary to the guide sequence, and hybridization between the target sequence and the guide sequence facilitates CRISPR complex formation. Complete complementarity is not necessarily required, as long as there is sufficient complementarity to induce hybridization and facilitate CRISPR complex formation. The target sequence may include any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be located within an organelle of a eukaryotic cell, for example, in a mitochondria or chloroplast.

[0089] When using multiple different guide sequences, a single expression construct may be used to target CRISPR activity to multiple different corresponding target sequences within the cell. For example, a single vector may contain about one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, or more guide sequences. In some embodiments, a vector containing about one, two, three, four, five, six, seven, eight, nine, ten, or more such guide sequences may be provided and optionally delivered to cells. In some embodiments, the vector includes a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein, also referred to as a Cas enzyme.

[0090] Non-exclusive examples of Cas proteins (or Cas enzymes) include Cas1, Cas1.13, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Cs Examples include m2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Crnr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs, or modified forms thereof.

[0091] These enzymes are known; for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2. In some embodiments, the CRISPR enzyme, e.g., Cas9, has DNA cleavage activity. In some embodiments, the CRISPR enzyme is Cas9, which may be Cas9 derived from S. pyogenes or S. pneumoniae.

[0092] In some embodiments, the CRISPR enzyme induces a cleavage of one or both strands at a location within the target sequence, such as within the target sequence and / or within a complementary sequence of the target sequence. In some embodiments, the CRISPR enzyme induces a cleavage of one or both strands within approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence.

[0093] Alternative modalities In some embodiments, the compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) comprises a small molecule. In some embodiments, the compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) comprises an activating peptide. In some embodiments, the compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) comprises an antibody.

[0094] Combination therapy In some embodiments, the present invention is characterized by a composition (e.g., one or more compositions, formulations, or drug formulations) or pharmaceutically acceptable combination comprising a compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) according to the present invention, and a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from one or more agents selected from the list consisting of clobazam, topiramate, prednisolone / prednisone, adrenocorticotropic hormone, and ganaxolone.

[0095] In some embodiments, the composition includes a pharmaceutically acceptable carrier. In some embodiments, the compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) according to the present invention, and the second agent, may exist in a single composition or as two or more different compositions. The compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) according to the present invention, and the second agent, may be administered via the same or different routes of administration. The compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) according to the present invention, and the second agent, may be administered simultaneously or sequentially. In some embodiments, the pharmaceutically acceptable combination includes the compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) according to the present invention and the second agent, separately or together.

[0096] Route of administration Compounds or compositions for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) according to the present invention, or pharmaceutical compositions according to the present invention, can be administered by different routes, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, buccal administration, intrathoracic, intravenous, intra-arterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal, and / or intra-articular, intraventricular injection, or combinations thereof. In some embodiments, the drug or pharmaceutical composition is administered orally. In some embodiments, the drug or pharmaceutical composition is administered intravenously. In some embodiments, the drug or pharmaceutical composition is administered by intraventricular injection. In some embodiments, the compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) according to the present invention, or pharmaceutical composition, is administered subcutaneously. Preferably, the compound or composition is administered in a manner that facilitates delivery to the brain of the target. Preferably, the compound or composition is administered such that the expression or activity of CDKL2 is increased in the brain of the target.

[0097] As described herein, the compounds and compositions of the present invention preferably increase the expression or activity of CDKL2 in the target brain. To facilitate such increase, the compounds or compositions may be formulated or delivered in a manner that facilitates delivery to the brain. For example, the compounds or compositions described herein may be formulated or delivered so that they cross the blood-brain barrier. The compounds or compositions described herein may be formulated or delivered so that they target cells of the CNS. One preferred approach is to utilize a modified AAV capsid protein that crosses the blood-brain barrier and targets CNS cells. For example, if the compound that increases CDKL2 is a nucleic acid molecule, the nucleic acid molecule may be delivered as a payload as part of a recombinant AAV particle. An alternative approach to achieve brain or CNS-specific delivery is to conjugate the compounds or compositions described herein with a targeting agent, e.g., a drug that targets the brain. The targeting agent may be a drug that crosses the blood-brain barrier. Such a drug (or conjugate) enables targeted drug delivery to the brain and is described (e.g.) in EP2308514. Such agents (or conjugates) may be particularly useful when the compound or composition that increases CDKL2 is a peptide, protein, or small molecule. The compounds or compositions described herein may be conjugated to a targeted ligand via a linker. [Examples]

[0098] Example 1 Figures 1–10 relate to rabbit anti-microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2) phospho-specific pS222 antibodies, produced using the same method as described in document

[29] .

[0099] Example 2 Figures 1, 2, and 3 relate to animal experiments. Mice were housed and treated in accordance with the regulations of the UK Animals (Scientific Treatment) Act 1986 and approved by the Francis Crick Institute Ethics Committee. Mice were housed, maintained in a 12-hour light / dark cycle, and given free access to food and water. Each mouse strain was reverse-crossed to a C57BL / 6J genetic background.

[0100] The Cdkl5 complete knockout mice were a generous donation from Cornelius Gross of the European Molecular Biology Laboratory in Rome.

[30] A Cdkl5 complete knockout mouse strain was maintained using heterozygous females and wild-type males, and only the brains of male offspring were taken to ensure Cdkl5 wild-type and hemizygous knockout littermates.

[0101] Residual pS222 EB2 was observed in CDKL5 KO(-Y) male mice compared to wild-type littermates (+ / Y), indicating that one or more different kinases can phosphorylate EB2 at Ser222 in addition to CDKL5 (Figure 1).

[0102] When CDKL5 is knocked out systemically (KO) or by a cell-type-specific driver, there is a reduction in CDKL5 levels (A) and pSer222 EB2 phosphorylation (B). See Figure 1A for the color coding of Cre drivers. The data show that CDKL5 and its activity (pEB2) are present in excitatory (green) and inhibitory (yellow and orange) neurons. CDKL5 activity was not observed in astrocytes (purple) (Figure 2).

[0103] The persistence of pS222 EB2 levels in the P8 cortex in systemic CDKL5 knockout mice indicates that another kinase / kinase can phosphorylate EB2. The absence of pS222 signaling in the cortex of EB2 phospho mutant mice confirms that no background is observed with the pS222 antibody used (Figure 3).

[0104] In rat primary neurons, 1 hour of phosphatase inhibitor treatment (okadaic acid / OA) induced an increase in pS222 EB2, indicating that this site is dephosphorylated by PP1 / PP2A phosphatases under normal conditions (Figure 4).

[0105] In primary cortical cultures prepared from CDKL5 knockout embryos, OA treatment increased phosphoryl-S222, indicating that kinases other than CDKL5 can phosphorylate this site. NMDA treatment reduced the pS222 signal, demonstrating that NMDA downregulates this phosphorylation and that the signal is not background (Figure 5).

[0106] Example 3 To generate Cdkl5 conditional knockout (cKO) mouse models by deleting CDKL5 in specific cell types, homozygous Cdkl5 phlox-positive females were mated with Cdkl5 heterozygous males expressing Nex-Cre for excitatory neurons, Dlx5 / 6-Cre or Vgat-Cre for inhibitory neurons, and Gfap-Cre for astrocytes. Brains were collected at P20 from Cdkl5 hemizygous phlox-positive males expressing Cre and Cre-negative littermates as controls (Figure 2). The Cdkl5 phlox-positive females were also donated by Cornelius Gross

[30] . The Nex-Cre mouse model (Neurod6tm1(cre)Kan, MGI:2668659) was generously donated by Dr. Klaus Nave. The Dlx5 / 6-Cre mouse model (Tg(mI56i-cre, EGFP)1Kc, MGI:3609985) was a generous donation from Dr. Francois Guillermot of the Francis Crick Institute. The Gfap-Cre mouse model (B6.Cg-Tg(Gfap-cre)77.6Mv, stock number:012887) was purchased from Jackson Laboratories. The Vgat-Cre (Slc32atm2(cre)Lowl / J, stock number:016962) was a generous donation from Dr. Andreas Schaefer of the Francis Crick Institute.

[0107] Example 4 Microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2) phospho mutant mice were generated using the CRISPR / Cas9 system by the Genetic Modification Service at the Francis Crick Institute. C57BL / 6J hyperovulatory females were mated with C57BL / 6J males, and then 25 ng / μL of guide RNA (gRNA), 100 ng of WT Cas9 mRNA, and 100 ng / μL of repair template were microinjected into the embryos. gRNA was generated using in vitro transcription (IVT). In short, annealed gRNA primers were ligated into a cloning vector, then IVT was performed on the gRNA plasmid using the MEGAshortscript kit (Ambion), and finally, the gRNA was purified using the MEGAclear kit (Ambion).

[0108] Two pairs of gRNA primers were designed and tested (Table 1), and one set of gRNA sequences was suitable for generating mutant mice. WT Cas9 mRNA was purchased from Tebubio. A repair template was ordered from Integrated DNA Technologies and had the following sequence: 5'- TTC TGC TTG CTT CTA GGC GCG GCT AAG TCA AGT CCT GCA TCT AAG CCA GGA TCC ACA CCT GCT CGC CCC GCG GCT GCC AAA AGG GCT TCA TCC AGT GGC TCT GCA TCC AGA TCT GAC AAA GAT TT A GAA ACG CAG GTC AT A CAG CTC AAC GAG CAG GTA ATT CAT CGC CTC CAC CCT TGT -3' (SEQ ID NO: 4). [Table 1]

[0109] Genomic DNA was extracted from ear clips, and the genotype of the variant was confirmed by amplifying the gRNA target site by PCR using the primers shown in Table 2.1. The resulting PCR products were washed with AMPure XP beads (Beckman Coulter) and sequenced using MiSeq (Illumina).

[0110] Example 5 Brain collection (Figures 1-3). For Western blotting. Fresh brains were rapidly collected from mice after cervical dislocation and decapitation. If necessary, brain regions were dissected and tissue was rapidly frozen in liquid nitrogen.

[0111] Example 6 - Cdkl5 WT and KO primary cultures Brains were collected from individual male E16.5 embryos from heterozygous Cdkl5 mothers and placed in hibernate buffer (Gibco), while Cdkl5 wild-type (WT) and KO genotypes were determined (Figures 4, 5, 6, 9, 10). Equal numbers of both genotypes were selected, and the cortex and hippocampus from these embryos were then dissected and washed three times individually with Hank equilibrium salt solution (HBSS). After incubation with 0.25% trypsin at 37°C for 15 minutes, the cells were washed four times with HBSS. Cells were dissociated and then counted using a hemocytometer. Neurons were directly plated on 18 mm glass coverslips (Fisher) or on plastic wells of 12-well culture plates at a density of 150,000–300,000 cells. Coverslips and wells were coated with 0.1 M borate buffer containing 60 μg / mL poly-D-lysine and 2.5 μg / mL laminin, and incubated overnight. Neurons were plated with minimal essential medium (MEM) containing 10% fetal bovine serum (FBS), 0.5% dextrose, 0.11 mg / mL sodium pyruvate, 2 mM glutamine, and penicillin / streptomycin. After 4 hours, the cultures were transferred to neuronal basal medium containing 1 mL of B27 (Gibco), 0.5 mM GlutaMAX, 0.5 mM glutamine, 12.5 μM glutamate, and penicillin / streptomycin. Primary neuronal cell cultures were maintained at 37°C and 5% CO2. 20–30% of the maintenance medium was replaced every 3–4 days.

[0112] Compared to CDKL5 KO cultures, primary cultures from male wild-type littermate control embryos showed significantly lower levels of pS222 EB2 in CDKL5 KO, but residual pS222 could still be upregulated by OA and downregulated by NMDA, indicating that some pS222 EB2 could persist and be regulated in CDKL5 KO (Figure 6).

[0113] Screening for other related kinases capable of phosphorylating pS222 EB2 showed that only CDKL5, ICK, and CDKL2 could phosphorylate EB2 (Figure 7). Further data showed that all three kinases could phosphorylate S222 on the known CDKL5 substrate EB2 in HEK293 cells (Figure 8) and primary cortical neurons (Figure 9).

[0114] Pregnant Long Evans rats were ordered from Jackson. E18.5 rat embryos were removed from the uterus, and the brains were extracted. The cortex was dissected, pooled from multiple animals, and washed three times with HBSS. The remainder of the protocol is identical to that used for mouse primary neurons (see Example 9).

[0115] Example 7 - HEK293T cells HEK293T cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with FBS and penicillin / streptomycin at 37°C and 5% CO2 for up to 25 passages (Figures 7–9). Each passage consisted of dissociating the cells using 0.25% trypsin-EDTA for 3 minutes at 37°C and adding a 1:10 ratio of cells to a new T75 flask. The remaining cells could be used for plating for future experiments. This step was repeated every 3–4 days. For all experiments, the cells were plated onto 12-well plates and transfected 24 hours after passage.

[0116] Example 8 The pLVExp-CMV-EGFP-rELAVI4 WT plasmid was purchased from VectorBuilder. The rELAVI4 phospho variant (SA) plasmid was generated by introducing the S131A mutation using site-directed mutagenesis (Figures 7-10). The pRK5-HA-CDKL51-352 (kinase domain) WT and kinase dead (KD) vectors, as well as the pTriex6-StrepII-EB2 vector, had already been described in

[29] . hCDKL1, hCDKL2, hCDKL3, hMAK, and hMOK were purchased from Addgene in the pDONR223 vector (#23389, #23421, #23628, 23624, and #23645, respectively) and transferred to the pCAG-DEST-3xFLAG-TwinStrep vector using gateway cloning (Invitrogen). hCDKL4 (NM_001346911.1) and hICK (NM_014920.4) were purchased in pcDNA3.1 / C-(K)-DYK vectors and transferred to pCAG-DEST-3xFLAG-TwinStrep vectors using the Gateway cloning method (Invitrogen). To generate kinase-dead plasmids, K33 34 36R for ICK, K33R for CDKL2, and K42R D153A for the CDKL5 mutation were generated using site-directed mutagenesis. The final plasmids were named ICK-FLAG KD, CDKL2-FLAG KD, and CDKL5-FLAG KD, respectively.

[0117] Example 9 - Neuron Culture Treatment Primary rat cortical neurons or Cdkl5 primary cortical neurons were plated onto 12-well plates and treated by adding the treatment directly to the culture medium (Figures 4-6). The neurons were then incubated at 37°C and lysed in 300 μL of 1X sample buffer (Invitrogen) containing 0.1 M DTT. Final concentrations and treatment durations are assay-dependent and are shown in the results section. The zero-time point is the control condition, where water or DMSO was added to the wells for the longest time point of the assay. For NMDA treatment in the absence of calcium, the neuronal culture medium was changed to artificial cerebrospinal fluid (ACSF) buffer (125 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2CO3, 25 mM glucose, 2 mM CaCl2, and 1 mM MgCl2) or Ca2+-free ACSF buffer (125 mM NaCl, 2.5 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2CO3, 25 mM glucose, 10 mM EGTA, and 1 mM MgCl2). The neurons were then treated and lysed in the same manner. For inhibition of synaptic NMDA receptors, the neurons were pre-incubated for 10 minutes with 10 μM MK-801 (Tocris) and 50 μM bicuculin (Tocris), then washed with fresh medium, treated with NMDA, and finally lysed.

[0118] CDKL2 was knocked down using primary neurons derived from the cortex of CDKL5 KO mice by lentiviral expression of shRNAs targeting CDKL2. Both shRNAs induced a reduction in the OA-dependent increase of pEB2 / EB2. The data suggest that CDKL2 is another kinase involved in EB2 phosphorylation in CDKL5 KO neurons (Figure 10).

[0119] Using the official Allen Brain Institute database, we analyzed single-cell mRNA expression of target kinases in adult human brain cells. ICK and CDKL4 expression are very low in human neurons. Therefore, CDKL2 is the most likely candidate for phosphorylating CDKL5 substrates such as EB2 in neurons (Figure 11).

[0120] Example 10 - Knockout Mouse CDKL5 / CDKL2 dual knockout mice were generated using the Crispr-Cas9 technique. Two distinct CDKL2 deletions were obtained, both lacking essential exon 4.

[0121] Using two pairs of guides: 5' guide: WGE ID:487701634 Guide 1-CACATAGCTTGAGAGCGTGC AGG (SEQ ID NO: 15), and WGE ID:487701635 Guide 2-ACATAGCTTGAGAGCGTGCA GGG (SEQ ID NO: 16), and 3' guide: WGE ID:487701575 Guide 3-ACGTCACCGACCAGCAGTTC TGG (SEQ ID NO: 17), and WGE ID:487701572 Guide 4-AAGTATGGCAAGTAAGCGGC AGG (SEQ ID NO: 18). Genomic deletions were confirmed by Sanger sequencing in primary mice.

[0122] We tested in vivo complementarity between CDKL5 and CDKL2 in mice. We assessed functional compensation by measuring pSer222 EB2 phosphorylation in proportion to total EB2. We hypothesized that if CDKL2 phosphorylates the CDKL5 target in vivo, the remaining EB2 phosphorylation would be reduced in the CDKL5 / CDKL2 double knockout compared to the CDKL5 single knockout.

[0123] Since CDKL5 activity is prominent throughout the whole brain, measurements were performed using whole brain lysates. CDKL5 homozygous knockout and CDKL2 heterozygous knockout mice were crossed to obtain different CDKL2 genotypes in the background of CDKL5 knockout mice. A dose-dependent reduction of the CDKL2 gene was observed at EB2 pSer222. These results indicate that, in vivo in mouse brain, CDKL2 is the primary compensatory kinase for CDKL5 activity, as measured by EB2 phosphorylation.

[0124] Patients with CDKL5 deficiency are expected to possess a functional CDKL2 gene. However, this is insufficient to compensate for the deficiency of CDKL5 in humans. These data further support the idea that increasing functional CDKL2 expression or activity in human neurons could be a treatment for CDKL5 deficiency.

[0125] Example 11 - CDKL5 / CDKL2 double knockout mice exhibit synergistic impairment in short-term memory. Methods: Adult mice were individually placed in a Y-shaped maze under low-light conditions, and their behavior was recorded for 10 minutes. The animals were used during the dark period of the light-dark cycle. Successful rotations within the maze were automatically calculated by Ethovision software.

[0126] Results: Since mice are expected to explore unvisited areas more frequently than recently visited areas, spontaneous alternation of arms within the Y-maze can be used as a measure of short-term working memory. Mice with good working memory remember previously visited arms in the maze and prefer to explore new arms, and fewer alternations indicate poorer working memory.

[0127] In this experiment, we compared wild-type (C57Bl / 6) mice with CDKL5 knockout (KO) mice and CDKL5 / 2 knockout mice. We hypothesized that if CDKL2 functions in parallel with CDKL5 in brain development, the combined loss of CDKL5 and CDKL2 would have a greater impact on brain function than the loss of CDKL5 alone. In the Y-maze, we found that CDKL2 / 5 dual knockout mice had a lower percentage of successful alternations compared to wild-type mice. There was no significant difference in successful alternations between CDKL5 KO mice and wild-type mice. These data suggest that CDKL2 and CDKL5 deletions together cause a decline in short-term memory, and that CDKL2 expression can compensate for the loss of CDKL5 function. These results provide further evidence that increasing CDKL2 could be used as a therapeutic option in CDKL5 deficiency.

[0128] Example 12: Increased CDKL2 in CDKL5-KO mice increases the phosphorylation of CDKL5 substrates. Methods: CDKL5-KO mice are treated with a drug that increases CDKL2 activity in the brain, or a control drug. The drug is a nucleic acid molecule, a viral vector encoding CDKL2, a small molecule, a polypeptide encoding CDKL2, or an antisense oligonucleotide. After treatment with the drug, the mouse brain is collected and the pSer222 EB2 phosphorylation level is evaluated by Western blotting or another method. Phosphorylation of other CDKL5 substrates, including MAP1S and CACNA1E, is also evaluated.

[0129] Results: CDKL5-KO mice exhibit reduced levels of EB2 phosphorylation compared to wild-type mice. Treatment of CDKL5-KO mice with a CDKL2-enhancing agent is expected to increase EB2 phosphorylation levels compared to CDKL5-KO mice treated with a control agent. Phosphorylation of other CDKL5 substrates, including MAP1S and CACNA1E, is also expected to increase in CDKL2-treated CDKL5-KO mice compared to CDKL5-KO mice treated with a control.

[0130] Example 13: Increased CDKL2 in CDKL5-KO mice alleviates behavioral disorders. Methods: CDKL5-KO mice were treated with a drug that increases CDKL2 activity in the brain, or with a control drug. The drug was either a nucleic acid molecule, a viral vector encoding CDKL2, a small molecule, a polypeptide encoding CDKL2, or an antisense oligonucleotide. Wild-type mice, mice treated with CDKL5-KO controls, and mice treated with CDKL5-KO CDKL2 activity enhancers were evaluated using behavioral assays including the Y-maze, field test, T-maze, fear memory, light / dark search, object recognition test, radial arm maze, hindlimb clasping, and Barnes maze.

[0131] Results: Mice treated with CDKL5-KO controls are expected to perform worse in behavioral assays compared to wild-type mice. Mice treated with CDKL5-KO CDKL2-enhancing agents are expected to perform better than mice treated with CDKL5-KO controls.

[0132] Summary of Results: CDKL5 deficiency (CDD) is caused by mutations in CDKL5 that lead to protein loss or reduced kinase activity and reduced phosphorylation of CDKL5 substrates. CDKL5 is X-linked. Due to X-chromosome inactivation, increasing CDKL5 gene expression (by targeting CDKL5 at the DNA or mRNA level) in cells expressing the CDKL5 gene, including loss of functional mutations, is not a viable therapeutic option for CDD (as it only increases the expression of the defective gene). As an alternative, we identified CDKL2 as a potential therapeutic target for CDD. The results above demonstrate that CDKL2 and CDKL5 are expressed in the same brain cell type and act on the same substrates. In particular, both CDKL5 and CDKL2 phosphorylate EB2 in neurons. Given this shared activity and the close systematic relationship between CDKL2 and CDKL5, increased CDKL2 levels in CDD compensate for the loss of CDKL5 function, restore normal neuronal activity in the CDD brain, and provide a viable therapeutic option for alleviating behavioral disorders.

[0133] Those skilled in the art will see that various modifications and variations can be made to the compositions and processes of the present invention. Accordingly, the present invention is intended to cover such modifications and variations, insofar as they fall within the scope of the appended claims and their equivalents.

[0134] All publications, patents, and patent applications referenced herein are incorporated herein by reference in whole to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated herein by reference. In addition, any citation or identification of references in this application should not be construed as an admission that such references are available as prior art of the present invention. Section headings should not necessarily be construed as restrictive to the extent in which they are used.

[0135] Further embodiments of the present invention are described below. 1. A compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2). 2. The compound or composition according to Embodiment 1, wherein the compound or composition comprises a nucleic acid molecule, such as a small interfering RNA (siRNA), a guide RNA (gRNA), or a short hairpin RNA (shRNA). 3. The compound or composition according to Embodiment 1 or 2, wherein the compound or composition comprises a nucleic acid molecule encoding a CDKL2 polypeptide. 4. The compound or composition according to Embodiment 3, wherein the nucleic acid molecule encoding CDKL2 encodes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the polypeptide of SEQ ID NO: 1. 5. The compound or composition according to Embodiment 3 or 4, wherein the nucleic acid molecule encoding CDKL2 encodes a polypeptide containing the sequence of Sequence ID No. 1. 6. The compound or composition according to any one of Embodiments 3 to 5, wherein the nucleic acid molecule encoding CDKL2 encodes a polypeptide consisting of the sequence of Sequence ID No. 1. 7. The compound or composition according to any one of Embodiments 3 to 6, wherein the nucleic acid molecule encoding CDKL2 contains a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with a nucleotide sequence selected from the list consisting of SEQ ID NO: 2 and SEQ ID NO: 3. 8. The compound or composition according to any one of Embodiments 3 to 7, wherein the nucleic acid molecule encoding CDKL2 comprises a nucleotide sequence selected from the list consisting of SEQ ID NO: 2 and SEQ ID NO: 3. 9. The compound or composition according to any one of Embodiments 3 to 8, wherein the nucleic acid molecule encoding CDKL2 consists of a nucleotide sequence selected from the list consisting of SEQ ID NO: 2 and SEQ ID NO: 3. 10. The compound or composition according to any one of Embodiments 1 to 9, wherein the compound or composition comprises a viral vector. 11. The compound or composition according to Embodiment 10, wherein the viral vector comprises one or more of the following: an adenovirus vector, an adeno-associated virus vector, a lentiviral vector, or a retroviral vector. 12. The compound or composition according to any one of Embodiments 2 to 11, wherein the nucleic acid molecule further comprises a promoter. 13. The compound or composition according to Embodiment 1 or 2, wherein the compound or composition comprises one or more clustered, regularly arranged short palindromic repeat (CRISPR)-related nucleases, transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZNFs). 14. The compound or composition according to any one of Embodiments 1, 2, or 13, comprising a CRISPR-related protein (Cas protein) and a guide RNA (gRNA) that hybridizes within the CDKL2 genome sequence. 15. The compound or composition according to Embodiment 14, wherein the CDKL2 genome sequence is defined by the Ensembl reference ENSG00000138769 (SEQ ID NO: 8). 16. The compound or composition according to Embodiment 1, wherein the compound or composition comprises an isolated CDKL2 polypeptide. 17. The compound or composition according to Embodiment 16, wherein the compound or composition comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% relative to the polypeptide of SEQ ID NO: 1. 18. The compound or composition according to Embodiment 16 or 17, wherein the compound or composition comprises the polypeptide of SEQ ID NO: 1. 19. The compound or composition according to any one of embodiments 16 to 18, wherein the compound or composition comprises the polypeptide of Sequence ID No. 1. 20. The compound or composition according to any one of the prior embodiments, wherein the compound or composition comprises or further comprises a small molecule activator for CDKL2 expression or activity. 21. The compound or composition according to any one of the prior embodiments, wherein the compound or composition comprises a small molecule activator that specifically binds to a protein structure as defined in the PDB database reference 4BBM. 22. The compound or composition according to any one of the prior embodiments, wherein the compound or composition comprises or further comprises a polypeptide that increases CDKL2 expression. 23. The compound or composition according to Embodiment 22, wherein the compound or composition comprises a transcription factor or enhancer that increases CDKL2 expression. 24. The compound or composition according to any one of the prior embodiments, wherein the compound or composition comprises or further comprises an antibody. 25. The compound or composition according to any one of the prior embodiments, wherein the compound or composition comprises a fusion protein containing a CDKL2 polypeptide operatively linked to a second polypeptide sequence. 26. The compound or composition according to Embodiment 25, wherein the second polypeptide sequence comprises a nuclear localization signal (NLS), a nuclear export signal (NES), a leader signal polypeptide, or a cell-permeable polypeptide. 27. A compound or composition according to any one of the prior embodiments, which increases CDKL2 activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% compared to CDKL2 activity measured in the absence of the compound or composition. 28. The compound or composition according to Embodiment 27, wherein CDKL2 activity is measured by the level of phosphorylation of the CDKL2 substrate and / or the level of CDKL2 autophosphorylation. 29. The compound or composition according to Embodiment 27 or 28, wherein CDKL2 activity is measured by the level of phosphorylation of microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2), microtubule-associated protein 1S (MAP1S), or voltage-gated R-type calcium channel subunit α-1E (CACNA1E). 30. The compound or composition according to Embodiment 29, wherein microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2) contains or consists of the amino acid sequence of SEQ ID NO: 5, microtubule-associated protein 1S (MAP1S) contains or consists of the amino acid sequence of SEQ ID NO: 6, or voltage-gated R-type calcium channel subunit α-1E (CACNA1E) contains or consists of the amino acid sequence of SEQ ID NO: 7. 31. The compound or composition according to any one of Embodiments 27 to 29, wherein CDKL2 activity is measured by the level of phosphorylation of microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2), and optionally, microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2) contains or comprises the amino acid sequence of SEQ ID NO: 5. 32. The compound or composition according to any one of the prior embodiments, which increases CDKL2 expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% compared to CDKL2 expression measured in the absence of the compound or composition. 33. The compound or composition according to Embodiment 32, wherein CDKL2 expression is measured by Western blotting, quantitative real-time PCR (qRT-PCR), advanced RNA sequencing, microarray, mass spectrometry, ELISA, or Phos-tag gel electrophoresis. 34. A compound or composition according to any one of the prior embodiments, further comprising a pharmaceutically acceptable carrier or excipient. 35. A compound or composition according to any one of the prior embodiments, further comprising a second therapeutic agent useful for treating CDKL5 deficiency. 36. The compound or composition according to Embodiment 35, wherein the second therapeutic agent is selected from the list consisting of clobazam, topiramate, prednisolone / prednisone, adrenocorticotropic hormone, and ganaxolone. 37. A method for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2), comprising administering a compound or composition described in any one of the prior embodiments to the target. 38. A method for treating or preventing a disease or disorder in a subject, comprising administering a compound or composition described in any one of Embodiments 1 to 36 to the subject. 39. A method for treating or preventing a neurological disorder or impairment in a subject, comprising administering a compound or composition described in any one of Embodiments 1 to 36 to the subject. 40. A method for treating or preventing cyclin-dependent kinase-like 5 (CDKL5) deficiency in a subject, comprising administering a compound or composition described in any one of Embodiments 1 to 36 to the subject. 41. A compound or composition according to any one of Embodiments 1 to 36 for use in a method for achieving the expression or increased activity of cyclin-dependent kinase-like 2 (CDKL2) in subjects requiring such expression or increased activity. 42. A compound or composition according to any one of Embodiments 1 to 36 for use in a method for treating or preventing a disease or disorder in a subject. 43. A compound or composition according to any one of Embodiments 1 to 36 for use in a method for treating or preventing a neurological disorder or impairment in a subject. 44. A compound or composition according to any one of Embodiments 1 to 36 for use in a method for treating or preventing cyclin-dependent kinase-like 5 (CDKL5) deficiency in a subject. 45. Use of a compound or composition according to any one of Embodiments 1 to 36 to achieve the expression or increased activity of cyclin-dependent kinase-like 2 (CDKL2) in subjects requiring such expression or increased activity. 46. ​​Use of any one of Embodiments 1 to 36 of a compound or composition for treating or preventing a disease or disorder in a subject. 47. Use of any one of Embodiments 1 to 36 of a compound or composition for the treatment or prevention of a neurological disorder or impairment in a subject. 48. Use of any one of the compounds or compositions described in Embodiments 1 to 36 for the treatment or prevention of cyclin-dependent kinase-like 5 (CDKL5) deficiency in a subject. 49. A method for use, compound or composition, or use according to any one of Embodiments 37 to 48, wherein the compound or composition is administered intrathecally, intravenously, intracystically, intraventricularly, or intraparum. 50. A method for use, compound or composition, or use according to any one of Embodiments 37 to 49, wherein the subject is a mammal. 51. A method for use, compound or composition, or use according to any one of Embodiments 37 to 50, wherein the subject is a human. 52. A method for use, compound or composition, or use according to Embodiment 50 or 51, wherein the compound or composition increases the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) in a subject. 53. A compound or composition according to any one of Embodiments 1 to 36 for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) in a subject. 54. A composition comprising a cyclin-dependent kinase-like 2 (CDKL2) activator. 55. The composition according to Embodiment 54, further comprising a pharmaceutically acceptable carrier or excipient. 56. The composition according to Embodiment 54 or 55, which increases CDKL2 activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% compared to CDKL2 activity measured in the absence of the composition. 57. The composition according to Embodiment 56, wherein CDKL2 activity is measured by the level of phosphorylation of the CDKL2 substrate and / or the level of CDKL2 autophosphorylation. 58. The composition according to Embodiment 56 or 57, wherein CDKL2 activity is measured by the level of phosphorylation of microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2), microtubule-associated protein 1S (MAP1S), or voltage-gated R-type calcium channel subunit α-1E (CACNA1E). 59. The composition according to Embodiment 58, wherein microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2) contains or consists of the amino acid sequence of SEQ ID NO: 5, microtubule-associated protein 1S (MAP1S) contains or consists of the amino acid sequence of SEQ ID NO: 6, or voltage-gated R-type calcium channel subunit α-1E (CACNA1E) contains or consists of the amino acid sequence of SEQ ID NO: 7. 60. The composition according to any one of embodiments 57 to 59, wherein CDKL2 activity is measured by the level of phosphorylation of microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2), and optionally, microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2) comprises or consists of the amino acid sequence of SEQ ID NO: 5. 61. The composition according to any one of embodiments 54 to 60, wherein the composition increases CDKL2 expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% compared to CDKL2 expression measured in the absence of the composition. 62. The composition according to Embodiment 61, wherein CDKL2 expression is measured by Western blotting, quantitative real-time PCR (qRT-PCR), advanced RNA sequencing, microarray, mass spectrometry, ELISA, or Phos-tag gel electrophoresis.

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[20] Mei,D.,C.Marini,F.Novara,B.D.Bernardina,T.Granata,E.Fontana,E.Parrini,A.R.Ferrari,A.Murgia,O.Zuffardi and R.Guerrini(2010).“Xp22.3 genomic deletions involving the CDKL5 gene in girls with early onset epileptic encephalopathy.”Epilepsia 51(4):647-654.

[21] Bartnik,M.,K.Derwinska,M.Gos,E.Obersztyn,K.E.Kolodziejska,A.Erez,A.Szpecht-Potocka,P.Fang,I.Terczynska,H.Mierzewska,N.J.Lohr,G.A.Bellus,T.Reimschisel,E.Bocian,T.Mazurczak,S.W.Cheung and P.Stankiewicz(2011).“Early-onset seizures due to mosaic exonic deletions of CDKL5 in a male and two females.”Genet Med 13(5):447-452.

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[24] https: / / www.rcsb.org / structure / 4BBM

[25] https: / / www.omim.org / entry / 300672

[26] https: / / www.icd10data.com / ICD10CM / Codes / G00-G99 / G40-G47 / G40- / G40.42

[27] Bahi-Buisson,N.,N.Villeneuve,E.Caietta,A.Jacquette,H.Maurey,G.Matthijs,H.Van Esch,A.Delahaye,A.Moncla,M.Milh,F.Zufferey,B.Diebold and T.Bienvenu(2012). “Recurrent mutations in the CDKL5 gene:genotype-phenotype relationships.” Am J Med Genet A 158a(7):1612-1619.

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[29] Baltussen,LL,Negraes,PD,Silvestre,M.,Claxton,S.,Moeskops,M.,Christodoulou,E.,Flynn,HR,Snijders,AP,Muotri,ARand Ultanir,SK,2018. Chemical genetic identification of CDKL 5 substrates reveals its role in neuronal microtubule dynamics. The EMBO Journal,37(24),p.e99763.

[30] Amendola E,Zhan Y,Mattucci C,Castroflorio E,Calcagno E,Fuchs C,Lonetti G,Silingardi D,Vyssotski AL,Farley D et al(2014)Mapping pathological phenotypes in a mouse model of CDKL5 disorder.PloS one 9:e91613

Claims

1. A compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) for use in methods of treating or preventing cyclin-dependent kinase-like 5 (CDKL5) deficiency in a subject.

2. The compound or composition for use according to claim 1, wherein the compound or composition comprises a nucleic acid molecule, a small interfering RNA (siRNA), a guide RNA (gRNA), or a short hairpin RNA (shRNA).

3. The compound or composition for use according to claim 1 or 2, wherein the compound or composition comprises a nucleic acid molecule encoding a CDKL2 polypeptide.

4. The compound or composition for use according to claim 3, wherein the nucleic acid molecule encoding CDKL2 encodes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the polypeptide of SEQ ID NO:

1.

5. The compound or composition for use according to claim 3 or 4, wherein the nucleic acid molecule encoding CDKL2 comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with a nucleotide sequence selected from the list consisting of SEQ ID NO: 2 and SEQ ID NO:

3.

6. The compound or composition for use according to any one of claims 3 to 5, wherein the nucleic acid molecule encoding CDKL2 comprises a nucleotide sequence selected from the list consisting of SEQ ID NO: 2 and SEQ ID NO:

3.

7. The compound or composition for use according to any one of claims 1 to 6, wherein the compound or composition comprises a viral vector.

8. The compound or composition for use according to claim 7, wherein the viral vector comprises one or more of the following: an adenovirus vector, an adeno-associated virus vector, a lentiviral vector, or a retrovirus vector.

9. The compound or composition for use according to any one of claims 2 to 8, wherein the nucleic acid molecule further comprises a promoter.

10. The compound or composition for use according to claim 1 or 2, wherein the compound or composition comprises one or more clustered, regularly arranged short palindromic repeat (CRISPR) related nucleases, transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZNFs).

11. The compound or composition for use according to any one of claims 1, 2, or 10, wherein the compound or composition comprises a CRISPR-related protein (Cas protein) and a guide RNA (gRNA) that hybridizes within the CDKL2 genome sequence.

12. The compound or composition for use according to claim 11, wherein the CDKL2 genome sequence is defined by Ensemble reference ENSG00000138769 (SEQ ID NO: 8).

13. The compound or composition for use according to claim 1, wherein the compound or composition comprises an isolated CDKL2 polypeptide.

14. The compound or composition for use according to claim 13, wherein the compound or composition comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% relative to the polypeptide of SEQ ID NO:

1.

15. The compound or composition for use according to any one of the prior claims, wherein the compound or composition comprises or further comprises a small molecule activator for CDKL2 expression or activity.

16. The compound or composition for use according to any one of the prior claims, wherein the compound or composition comprises or further comprises a polypeptide that increases CDKL2 expression.

17. A compound or composition for use according to any one of the prior claims, which increases the CDKL2 activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% compared to the CDKL2 activity measured in the absence of the compound or composition.

18. The compound or composition for use according to claim 17, wherein CDKL2 activity is measured by the level of phosphorylation of the CDKL2 substrate and / or the level of CDKL2 autophosphorylation.

19. A compound or composition for use according to claim 17 or 18, wherein CDKL2 activity is measured by the level of phosphorylation of microtubule-associated protein RP / EB family member 2 (MAPRE2 / EB2), microtubule-associated protein 1S (MAP1S), or voltage-gated R-type calcium channel subunit α-1E (CACNA1E).

20. A compound or composition for use according to any one of the prior claims, wherein the compound or composition increases CDKL2 expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% compared to the CDKL2 expression measured in the absence of the compound or composition.

21. The compound or composition for use according to claim 20, wherein CDKL2 expression is measured by Western blotting, quantitative real-time PCR (qRT-PCR), advanced RNA sequencing, microarray, mass spectrometry, ELISA, or Phos-tag gel electrophoresis.

22. A compound or composition for use according to any one of the prior claims, further comprising a pharmaceutically acceptable carrier or excipient.

23. A compound or composition according to any one of claims 1 to 22, for use in a method for treating or preventing neurological diseases or disorders in a subject.

24. A compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2) in the target brain.

25. Recombinant adeno-associated virus (AAV) particles comprising an AAV (adeno-associated virus) capsid protein and a nucleic acid payload, wherein the nucleic acid payload encodes a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the polypeptide of SEQ ID NO: 1, and the recombinant AAV particles are capable of crossing the blood-brain barrier.

26. Recombinant adeno-associated virus (AAV) particles comprising an AAV capsid protein and a nucleic acid payload, wherein the nucleic acid payload encodes the polypeptide of Sequence ID No. 1, and the recombinant AAV particles are capable of crossing the blood-brain barrier.

27. Recombinant adeno-associated virus (AAV) particles comprising an AAV capsid protein and a nucleic acid payload, wherein the nucleotide sequence has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with a nucleotide sequence selected from the list consisting of SEQ ID NOs: 2 and SEQ ID NOs: 3, and the recombinant AAV particles are capable of crossing the blood-brain barrier.

28. Recombinant adeno-associated virus (AAV) particles comprising an AAV capsid protein and a nucleic acid payload, wherein the nucleic acid payload comprises a nucleotide sequence selected from the list consisting of SEQ ID NOs: 2 and SEQ ID NOs: 3, and the recombinant AAV particles are capable of crossing the blood-brain barrier.

29. A compound or composition for increasing the expression or activity of cyclin-dependent kinase-like 2 (CDKL2), wherein the compound or composition is conjugated with a targeting agent.

30. The compound or composition according to claim 29, wherein the targeting agent is a drug that targets the brain.

31. The compound or composition according to claim 29 or 30, wherein the targeting agent is a drug that crosses the blood-brain barrier.

32. The compound or composition according to any one of claims 29 to 31, wherein the compound or composition is a peptide, protein, or small molecule.

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