Patent Application
20230043046
The present invention generally relates to the treatment of kinase deficiency disorders, particularly novel recombinant proteins and gene therapy for the treatment of disorders involving deficiency of CDKL5.
CDKL5 is a serine/threonine kinase and was previously known as STK9. Mutations in this gene have recently been associated with a number of neurological disorders such as mental retardation, loss of communication and motor skills, infantile spasms and seizures, atypical Rett Syndrome, and X-linked West Syndromes. Mutations or deletions of the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been shown to cause an epileptic encephalopathy with early-onset severe neurological impairment and intractable seizures.
Currently, the oldest known people described in medical literature with CDKL5 deficiency have reached an age of 41 years old. Many others are in their twenties and teens, but because the disease has only been identified in the last 15 years, the majority of newly diagnosed are toddlers or infants. Individuals diagnosed with CDKL5 deficiency disorder generally suffer delays in neurological development and are at a high risk for seizures, with a median onset age of 6 weeks. One study of 111 participants found that 85.6% of individuals had epilepsy with a daily occurrence of seizures, and a mean of 6 seizures per day.
Current treatments range from seizure medications, ketogenic diets, vagal nerve stimulation, and surgery. Commonly administered anti-epileptic medications include clobazam, valproic acid, and topiramate, and in many cases two or more medication regiments are used at the same time. Individuals seemed to have a “honeymoon period” in which they are seizure free for a period of time after starting a new type of medication, but ultimately there is a recurrence of seizures. The duration of observed honeymoon ranges from 2 months to 7 years, with a median of 6 months. For example, the study found that 16 of the 111 participants were currently seizure free, and one individual had never developed seizures.
The exact mechanisms for pathogenic manifestations remain unclear. Some experimental data suggest that certain non-sense mutations in the C-terminus cause the protein to be constitutively localized to the nucleus, while other missense mutations are highly represented in the cytoplasm. Nuclear localization signals and nuclear export signals have both been identified in the C-terminus of the protein.
Some mutant enzyme variants result in partial or total loss of phosphorylation function, while other mutations and truncations result in an increase in phosphorylation capacity, suggesting that both loss and gain of function may be pathogenic. Interactions and pathogenic effects arising from enzymatic activity loss/gain of function and enzyme nuclear localization versus residence in the cytoplasm remain unclear. An analysis of patients with a wide range of CDKL5 mutations and presenting clinical symptoms suggests that mutations causing clinical symptoms are more likely to be found either in the C-terminus or the kinase activity domain, suggesting that both the kinase activity and protein translocation capacity of CDKL5 could affect the clinical manifestation of symptoms.
Accordingly, various aspects of the invention pertain to new recombinant CDKL5 proteins and gene therapy compositions, which can be used to treat CDKL5-mediated neurological disorders such as a CDKL5 deficiency or an atypical Rett syndrome caused by a CDKL5 mutation or deficiency. Other aspects of the invention pertain to methods of producing such recombinant CDKL5 proteins and gene therapy compositions, as well as pharmaceutical compositions, methods of treatment, and uses of such recombinant proteins and gene therapy compositions.
One aspect of the present invention relates to a composition comprising a gene therapy delivery system and a CDKL5 polynucleotide encoding a CDKL5 polypeptide. In various embodiments, the CDKL5 polypeptide has at least 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.
In one or more embodiments, the CDKL5 polypeptide has at least 98% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 26. In one or more embodiments, the CDKL5 polynucleotide has at least 90% sequence identity to SEQ ID NO: 123.
In one or more embodiments, the CDKL5 polypeptide has at least 98% sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.
In one or more embodiments, the CDKL5 polypeptide has at least 98% sequence identity to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25. In one or more embodiments, the CDKL5 polynucleotide has at least 90% sequence identity to SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147 or 1 SEQ ID NO: 149.
In one or more embodiments, the gene therapy delivery system comprises one or more of a viral vector, a liposome, a lipid-nucleic acid nanoparticle, an exosome and a gene editing system. In one or more embodiments, the gene editing system comprises one or more of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) associated protein 9 (CRISPR-Cas-9), Transcription activator-like effector nuclease (TALEN) or ZNF (Zinc finger protein).
In one or more embodiments, the gene therapy delivery system comprises a viral vector. In one or more embodiments, the viral vector comprises one or more of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, a poxviral vector or a herpes simplex viral vector. In one or more embodiments, the viral vector comprises a viral polynucleotide operably linked to the CDKL5 polynucleotide. In one or more embodiments, the viral vector comprises at least one inverted terminal repeat (ITR).
In one or more embodiments, the composition further comprises one or more of an SV40 intron, a polyadenylation signal or a stabilizing element.
In one or more embodiments, the composition further comprises a promoter. In one or more embodiments, the promoter has at least 90% sequence identity to SEQ ID NO: 29 or SEQ ID NO: 30.
In one or more embodiments, the composition further comprises a polynucleotide encoding a cell-penetrating polypeptide. In one or more embodiments, the cell-penetrating polypeptide has at least 90% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 167. In one or more embodiments, the polynucleotide encoding the cell-penetrating peptide has at least 90% sequence identity to SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172 or SEQ ID NO: 173.
In one or more embodiments, the composition further comprises a polynucleotide encoding a leader signal polypeptide. In one or more embodiments, the leader signal polypeptide has at least 90% sequence identity to SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166 or SEQ ID NO: 168. In one or more embodiments, the polynucleotide encoding the leader signal polypeptide has at least 90% sequence identity to SEQ ID NO: 155. In one or more embodiments, the polynucleotide encoding the leader signal polypeptide has at least 90% sequence identity to SEQ ID NO: 169.
Another aspect of the present invention relates to a pharmaceutical formulation comprising a composition as described herein and a pharmaceutically acceptable carrier.
Another aspect of the present invention relates to a method of treating a CDKL5-mediated neurological disorder, the method comprising administering a composition or formulation as described herein to a patient in need thereof. In one or more embodiments, the composition or the formulation is administered intrathecally, intravenously, intracistnerally, intracerebroventrically or intraparenchymally. In one or more embodiments, the CDKL5-mediated neurological disorder is one or more of a CDKL5 deficiency or an atypical Rett syndrome caused by a CDKL5 mutation or deficiency.
Another aspect of the present invention relates to a method of treating a CDKL5-mediated neurological disorder, the method comprising administering a composition or formulation as described herein to an ex vivo cell and administering the ex vivo cell to a patient in need thereof. In one or more embodiments, ex vivo cell is administered intrathecally, intravenously, intracistnerally, intracerebroventrically or intraparenchymally. In one or more embodiments, the CDKL5-mediated neurological disorder is one or more of a CDKL5 deficiency or an atypical Rett syndrome caused by a CDKL5 mutation or deficiency.
Another aspect of the present invention relates to a novel CDKL5 polypeptide. In various embodiments, the CDKL5 polypeptide comprises a sequence having at least 99% sequence identity to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25. In one or more embodiments, the CDKL5 polypeptide comprises the sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25. In one or more embodiments, the CDKL5 polypeptide comprises the sequence of SEQ ID NO: 13. In one or more embodiments, the CDKL5 polypeptide comprises the sequence of SEQ ID NO: 14. In one or more embodiments, the CDKL5 polypeptide comprises the sequence of SEQ ID NO: 15. In one or more embodiments, the CDKL5 polypeptide comprises the sequence of SEQ ID NO: 16. In one or more embodiments, the CDKL5 polypeptide comprises the sequence of SEQ ID NO: 17. In one or more embodiments, the CDKL5 polypeptide comprises the sequence of SEQ ID NO: 18. In one or more embodiments, the CDKL5 polypeptide comprises the sequence of SEQ ID NO: 19. In one or more embodiments, the CDKL5 polypeptide comprises the sequence of SEQ ID NO: 20. In one or more embodiments, the CDKL5 polypeptide comprises the sequence of SEQ ID NO: 21. In one or more embodiments, the CDKL5 polypeptide comprises the sequence of SEQ ID NO: 22. In one or more embodiments, the CDKL5 polypeptide comprises the sequence of SEQ ID NO: 23. In one or more embodiments, the CDKL5 polypeptide comprises the sequence of SEQ ID NO: 24. In one or more embodiments, the CDKL5 polypeptide comprises the sequence of SEQ ID NO: 25.
Another aspect of the present invention relates to a fusion protein comprising a CDKL5 polypeptide as described herein and a leader signal polypeptide operatively coupled to the CDKL5 polypeptide. In one or more embodiments, the leader signal polypeptide has at least 90% sequence identity to SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 or SEQ ID NO: 168. In one or more embodiments, the leader signal polypeptide comprises the sequence of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 or SEQ ID NO: 168.
Another aspect of the present invention relates to a fusion protein comprising a CDKL5 polypeptide as described herein and a cell-penetrating polypeptide operatively coupled to the CDKL5 polypeptide. In one or more embodiments, the cell-penetrating polypeptide has at least 90% sequence identity to SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 167. In one or more embodiments, the cell-penetrating polypeptide comprises the sequence of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 167. In one or more embodiments, the fusion protein further comprises a leader signal polypeptide. In one or more embodiments, the leader signal polypeptide has at least 90% sequence identity to SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 or SEQ ID NO: 168. In one or more embodiments, the leader signal polypeptide comprises the sequence of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 or SEQ ID NO: 168.
In one or more embodiments, the fusion protein further comprises one or more affinity-tags, one or more protease cleavage sites, or combinations thereof. In some embodiments, the affinity-tag comprises one or more of MYC, HA, V5, NE, StrepII, Twin-Strep-tag®, glutathione S-transferase (GST), maltose-binding protein (MBP), calmodulin-binding peptide (CBP), FLAG®, 3×FLAG®, polyhistidine (His), HPC4, or combinations thereof. In some embodiments, the protease cleavage site is sensitive to one or more of thrombin, furin, factor Xa, metalloproteases, enterokinases, cathepsin, HRV3C, TEV, or combinations thereof.
Another aspect of the present invention relates to a pharmaceutical formulation comprising a CDKL5 polypeptide or fusion protein as described herein and a pharmaceutically acceptable carrier.
Another aspect of the present invention relates to a method of treating a CDKL5-mediated neurological disorder, the method comprising administering a CDKL5 polypeptide or fusion protein or formulation as described herein to a patient in need thereof. In one or more embodiments, the polypeptide, fusion protein or formulation is administered intrathecally, intravenously, intracisternally, intracerebroventrically or intraparenchymally. In one or more embodiments, the CDKL5-mediated neurological disorder is one or more of a CDKL5 deficiency or an atypical Rett syndrome caused by a CDKL5 mutation or deficiency.
Another aspect of the present invention relates to a method of producing a CDKL5 polypeptide or fusion protein as described herein. In various embodiments, the method comprises expressing the CDKL5 polypeptide or the fusion protein and purifying the CDKL5 polypeptide or the fusion protein. In one or more embodiments, the CDKL5 polypeptide or the fusion protein is expressed in Chinese hamster ovary (CHO) cells, HeLa cells, human embryonic kidney (HEK) cells or Escherichia coli cells.
Another aspect of the present invention relates to a method of producing a protein comprising a CDKL5 polypeptide, the method comprising expressing the protein in insect cells and purifying the protein from the insect cells. In one or more embodiments, the insect cells are Sf9 cells or BTI-Tn-5B1-4 cells.
In one or more embodiments, the protein comprises a fusion protein comprising the CDKL5 polypeptide and a cell-penetrating polypeptide operatively coupled to the CDKL5 polypeptide. In one or more embodiments, the cell-penetrating polypeptide is operatively coupled to the N-terminus of the CDKL5 polypeptide. In one or more embodiments, the cell-penetrating polypeptide is operatively coupled to the C-terminus of the CDKL5 polypeptide. In one or more embodiments, the fusion protein further comprises a leader signal polypeptide.
In one or more embodiments, the fusion protein further comprises one or more affinity-tags, one or more protease cleavage sites, or combinations thereof. In some embodiments, the affinity-tag comprises MYC, HA, V5, NE, StrepII, Twin-Strep-tag®, glutathione S-transferase (GST), maltose-binding protein (MBP), calmodulin-binding peptide (CBP), FLAG®, 3×FLAG®, polyhistidine (His), HPC4, or combinations thereof. In some embodiments, the protease cleavage site is sensitive to one or more of thrombin, furin, factor Xa, metalloproteases, enterokinases, cathepsin, HRV3C, TEV, or combinations thereof.
In one or more embodiments, the CDKL5 polypeptide has at least 98% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26. In one or more embodiments, the CDKL5 polypeptide has at least 98% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 26. In one or more embodiments, the CDKL5 polypeptide has at least 98% sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Before describing several exemplary embodiments of the invention, it is to be understood that the invention is not limited to the details of construction or process steps set forth in the following description. The invention is capable of other embodiments and of being practiced or being carried out in various ways.
It has surprisingly been discovered that proteins comprising the wild-type CDKL5 sequence have significant N-linked glycosylation when expressed and secreted in various host cell systems. Such N-linked glycosylation may have a negative impact on enzyme function due to changes in folding and/or interactions with binding partners. Accordingly, various aspects of the present invention relate to recombinant proteins comprising CDKL5 polypeptides that have one or more mutations to remove N-linked glycosylation sites.
Moreover, without wishing to be bound by any particular theory, it is believed that shorter CDKL5 variants that retain functional activity can provide benefits over the full-length, wild-type CDKL5 polypeptide, particularly when incorporated into a fusion protein comprising the CDKL5 polypeptide. In one or more embodiments, such benefits can include improved secretion from host cells during protein production, improved solubility, enhanced ability to cross the blood-brain barrier (BBB), and/or enhanced ability to penetrate target cells.
Other aspects of the present invention relate to novel cell systems for expressing and secreting recombinant proteins comprising CDKL5 polypeptides (e.g., wild-type CDKL5 polypeptides, CDKL5 variants with one or more N-linked glycosylation sites removed and/or shorter CDKL5 variants).
Other aspects of the present invention relate to gene therapy compositions and methods that utilize a CDKL5 polynucleotide encoding a CDKL5 polypeptide as described herein and a gene therapy delivery system.
As used herein, the term “CDKL5-mediated neurological disorder” refers to any disease or disorder that can be treated by expression or overexpression of the CDKL5 protein.
As used herein, the term “CDKL5 deficiency” refers to any deficiency in the biological function of the protein. The deficiency can result from any DNA mutation in the DNA coding for the protein or a DNA related regulatory region or any change in the function of the protein due to any changes in epigenetic DNA modification, including but not limited to DNA methylation or histone modification, any change in the secondary, tertiary, or quaternary structure of the CDKL5 protein, or any change in the ability of the CDKL5 protein to carry out its biological function as compared to a wild-type or normal subject. The deficiency can also include a lack of CDKL5 protein, such as a null mutation or underexpression of a fully functioning protein.
As used herein, the term “atypical Rett syndrome caused by a CDKL5 mutation or deficiency” refers to an atypical form of Rett syndrome with similar clinical signs to Rett syndrome but is caused by a CDKL5 mutation or deficiency.
Symptoms or markers of a CDKL5 deficiency, Rett syndrome, or an atypical Rett syndrome include but are not limited to seizures, cognitive disability, hypotonia, as well as autonomic, sleep, and gastrointestinal disturbances.
As used herein, the term “gene therapy delivery system” refers to any system that can be used to deliver an exogenous gene of interest to a target cell so that the gene of interest will be expressed or overexpressed in the target cell. In one or more embodiments, the target cell is an in vivo patient cell. In one or more embodiments, the target cell is an ex vivo cell and the cell is then administered to the patient.
As used herein, the term “carrier” is intended to refer to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Suitable pharmaceutical carriers are known in the art and, in at least one embodiment, are described in “Remington's Pharmaceutical Sciences” by E. W. Martin, 18th Edition, or other editions.
As used herein, the term “enzyme replacement therapy” or “ERT” is intended to refer to the introduction of an exogenous, purified enzyme into an individual having a deficiency in such enzyme. The administered protein can be obtained from natural sources or by recombinant expression. The term also refers to the introduction of a purified enzyme in an individual otherwise requiring or benefiting from administration of a purified enzyme. In at least one embodiment, such an individual suffers from enzyme insufficiency. The introduced enzyme may be a purified, recombinant enzyme produced in vitro, or a protein purified from isolated tissue or fluid, such as, for example, placenta or animal milk, or from plants.
As used herein, the terms “subject” or “patient” are intended to refer to a human or non-human animal. In at least one embodiment, the subject is a mammal. In at least one embodiment, the subject is a human.
As used herein, the “therapeutically effective dose” and “effective amount” are intended to refer to an amount of gene therapy composition (e.g. comprising CDKL5 polynucleotides) or recombinant protein (e.g. CDKL5 variants or fusion proteins) which is sufficient to result in a therapeutic response in a subject. A therapeutic response may be any response that a user (for example, a clinician) will recognize as an effective response to the therapy, including any surrogate clinical markers or symptoms described herein and known in the art. Thus, in at least one embodiment, a therapeutic response can be an amelioration or inhibition of one or more symptoms or markers of a CDKL5 deficiency, Rett syndrome, or an atypical Rett syndrome such as those known in the art.
The human CDKL5 gene is composed of 24 exons, of which the first three (exons 1, 1a and 1b) are untranslated.
The originally discovered human CDKL5 variant was 1030 amino acids with a molecular mass of 115 kDa (CDKL5115). Another prominent variant, CDKL5107, contains an altered C-terminal region because alternative splicing combines different exons than in the CDKL5115 variant. CDKL5107 (107 kDa) is shorter because it harbors an alternate version of exon 19 and does not contain exons 20-21 that are present in the CDKL5115 variant. The hCDKL5107 mRNA has been found to be 37-fold more abundant in human brain than the hCDKL5115 transcript, and murine CDKL5107 has been found to be 160-fold more abundant than the murine CDKL5105 variant in murine brain. Both the human and murine CDKL5107 isoforms have demonstrated a longer half-life and resistance to degradation as compared to the human CDKL5115 variant.
CDKL5 knockout mouse models have been generated using the Lox-Cre recombination system and these mice present symptoms of autistic-like deficits in social interactions, impairment of motor control, and loss of fear memory (Wang et al., Proc Natl Acad Sci U.S.A, 109(52), 21516-21521). For example, knockout CDKL5 mice have symptoms of reduced motor coordination and demonstrate impaired memory and fear responses when repeatedly exposed to stimuli. These changes have led scientists to hypothesize that loss of CDKL5 kinase activity leads to impaired neuronal network development. Previous data have suggested that CDKL5 phosphorylates methyl-CpG binding protein 2 (MeCP2), and independent loss-of-function mutations in MeCP2 lead to the Rett syndrome phenotype. Other substrates of CDKL5 include Netrin G1 ligand (NGL-1), Shootin1 (SHTN1), Mindbomb 1 (MIB1), DNA (cytosine-5)-methyltransferase 1 (DNMT1), Amphiphysin 1 (AMPH1), end-binding protein EB2, microtubule associated protein 1S (MAP1S) and histone deacetylase 4 (HDAC4). Although the exact role of CDKL5 has yet to be identified, these data suggest that CDKL5 plays a role in phosphorylation of downstream targets that are critical for correct neuronal development, including MeCP2. In humans, mutations in CDKL5 are associated with a phenotype that overlaps with Rett syndrome, and additionally presents with early-onset seizures. While CDKL5 KO mice did not exhibit any early-onset seizure symptoms, they did exhibit motor defects, decreased sociability, and impaired learning and memory (Chen et al. CDKL5, a protein associated with Rett Syndrome, regulates neuronal morphogenesis via Rac1 signaling, J Neurosci 30: 12777-12786).
Two CDKL5 isoforms are found in rat, one labeled CDKL5a and the other CDKL5b. (Chen et al.). In general, there is a high level of sequence conservation in CDKL5 genes across human, rat, and mouse species except for the last 100-150 amino acids near the C-terminus. Western blot data show that both variants are present during rat development yet adults appear to predominately express a single variant. Furthermore, CDKL5 is present in identifiable quantities in brain, liver, and lung.
CDKL5 functions in the nucleus but it is also found in the dendrites of cultured neurons, suggesting a possible alternate cytoplasmic role. Down regulation of CDKL5 expression by RNAi (RNA Interference) in cultured cortical neurons inhibited neurite growth and dendritic arborization (branching), where over expression of CDKL5 had opposite effects (Chen et al.). In order to characterize both the nuclear and cytoplasmic effect of CDKL5, a variant of CDKL5a with a nuclear export sequence (NES) was expressed in the cultured cortical neuron RNAi model. This NES-CDKL5a variant was resistant to the RNAi used to silence the wild-type gene expression, and therefore was used to model CDKL5a when expressed solely in the cytoplasm. After using the GFP tag to confirm that this CDKL5 variant was exclusively present in the cytoplasm, an increase in both the length of neurites and number of neurite branches was seen. The ability of NES-GFP-CDKL5a to partially rescue the disease phenotype observed when RNAi was used to knockdown the endogenous CDKL5 expression suggests that the expression of CDKL5 in cytoplasm in an important factor in the development and growth of neurites.
Human mutations in CDKL5 are associated with a phenotype similar to Rett syndrome, and individuals with CDKL5 mutations also present with early-onset seizures. This onset of seizures differs from the classical Rett syndrome phenotype in which there is an early normal period of development before the onset of Rett symptoms. Patients with classical Rett syndrome (RTT) appear to develop normally until 6-18 months of age, and then they begin to present neurological symptoms including loss of speech and movement. Autopsies of RTT brains show smaller and more densely packed neurons with shorter dendrites in the motor and frontal cortex, suggesting that neuronal development is impaired. The majority of Classical RTT cases are due to mutations in the MECP2 gene, which is an X-linked gene encoding a nuclear protein that selectively binds to CpG dinucleotides in the mammalian genome and regulates transcription through the recruitment of complexes. Although poorly understood, it is generally thought that the dysregulation of gene expression caused by mutations in MECP2 is the underlying cause of Rett Syndrome. Approximately 20% of Classic Rett syndrome cases and 60-80% of other Rett syndrome variants carry no mutations in MECP2, suggesting an alternate genetic cause for pathogenesis. Recently, some CDKL5 mutations have been identified in patients with certain variants of RTT and other severe encephalopathies, and CDKL5 has been shown to interact with MeCP2 both in vivo and in vitro. Beyond MeCP2, CDKL5 has been shown to interact with and phosphorylate a number of downstream targets, including NGL-1. When phosphorylated, NGL-1 interacts with PSD95 and is critical for the correct genesis and development of dendritic spines and synapse formation (Ricciardi S, et al. “CDKL5 ensures excitatory synapse stability by reinforcing NGL-1-PSD95 interaction in the postsynaptic compartment and is impaired in patient iPSC-derived neurons.” Nat Cell Biol 14(9):911-923).
CDKL5 has also been shown to phosphorylate the protein DNA methyltransferase 1 (DNMT1) (Kameshita I, et al. “Cyclin-dependent kinase-like 5 binds and phosphorylates DNA methyltransferase 1.” Biochem Biophys Res Commun 377:1162-1167). This phosphorylation leads to activation of DNMT1, which is a maintenance-type methylation protein that preferentially methylates hemimethylated DNA. This process is useful for maintenance of DNA methylation patterns during DNA replication, so that newly synthesized daughter DNA strands are able to maintain the methylation pattern of the parent strand it replaced. As methylation of DNA is generally thought to be an epigenetic mechanism to silence gene expression, this maintenance function of DNMT1 is crucial in preserving gene expression patterns across cell generations.
Current models suggest that the CDKL5 kinase domain phosphorylates GSK-3β, and that phosphorylation of GSK-3β leads to its inactivation. Individuals who are deficient in CDKL5 activity therefore seem to exhibit increased GSK-3β activity. Previous studies have shown that GSK-3β modulates hippocampal neurogenesis, and that an increased activity of GSK-3β severely impairs dendritic morphology of newborn hippocampal neurons. Furthermore, GSK-3β seems to act as a negative regulator of key developmental events such as neuron survival and maturation. A study conducted using CDKL5 KO mice demonstrated that treatment with a GSK-3β inhibitor could almost fully rescue hippocampal development and behavioral deficits in mice deficient in CDKL5 activity (Fuchs et al. “Inhibition of GSK3β Rescues Hippocampal Development and Learning in a Mouse Model of CDKL5 Disorder.” Neurobiology of Disease 82: 298-310). This developmental rescue also seemed to persist beyond treatment.
Various embodiments of the present invention provide novel CDKL5 variants.
In one or more embodiments, the CDKL5 polypeptide has at least 98%, at least 98.5%, at least 99% or at least 99.5% sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. The CDKL5 polypeptide may contain deletions, substitutions and/or insertions relative to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more deletions, substitutions and/or insertions to the amino acid sequence described by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.
In one or more embodiments, the CDKL5 polypeptide has at least 98%, at least 98.5%, at least 99% or at least 99.5% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 26. The CDKL5 polypeptide may contain deletions, substitutions and/or insertions relative to SEQ ID NO: 1 or SEQ ID NO: 26, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more deletions, substitutions and/or insertions to the amino acid sequence described by SEQ ID NO: 1 or SEQ ID NO: 26.
In one or more embodiments, the CDKL5 polypeptide comprises one or more affinity-tags. In one or more embodiments, the affinity-tag is located on one or more of the N-terminus or the C-terminus of the CDKL5 polypeptide. Examples of tags that can be added to the fusion proteins include, but are not limited to, epitope tags (e.g. MYC, HA, V5, NE, StrepII, Twin-Strep-tag®, HPC4), glutathione S-transferase (GST), maltose-binding protein (MBP), calmodulin-binding peptide (CBP), FLAG®, 3×FLAG®, polyhistidine (His), and combinations thereof.
In one or more embodiments, the CDKL5 polypeptide comprises one or more protease cleavage sites. In some embodiments, the protease cleavage site is located on one or more of the N-terminus or the C-terminus of the CDKL5 polypeptide. Exemplary protease cleavage sites include, but are not limited to, cleavage sites sensitive to thrombin, furin, factor Xa, metalloproteases, enterokinases, cathepsin, HRV3C, TEV, and combinations thereof.
Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting. For example, polypeptides having at least 98%, 98.5%, 99% or 99.5% identity to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotide encoding such polypeptides, are contemplated. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score. BLASTP “Identities” shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP “Positives” shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity of similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. The polynucleotide sequences of similar polypeptides are deduced using the genetic code and may be obtained by conventional means, in particular by reverse translating its amino acid sequence using the genetic code.
One skilled in the art can readily derive a polynucleotide sequence encoding a particular polypeptide sequence. Such polynucleotide sequence can be codon optimized for expression in the target cell using commercially available products, such as using the OptimumGene™ codon optimization tool (GenScript, Piscataway, N.J.).
Various embodiments of the present invention provide novel CDKL5 variants that have one or more mutations to remove one or more N-linked glycosylation sites from the CDKL5 polypeptide. The wild-type human isoform CDKL5107 contains 10 potential N-linked glycosylation sites and the wild-type human isoform CDKL5115 contains 8 potential N-linked glycosylation sites. One of these glycosylation sites includes the TEY (Thr-Glu-Tyr) motif: NYTEY (Asn-Tyr-Thr-Glu-Tyr), and thus one of the glycosylation sites resides in the kinase domain. As such, there is a high likelihood that glycosylation at the Asn-Tyr-Thr-Glu-Tyr site can interfere with phosphorylation of the Thr-Glu-Tyr motif. Generally, sequences of Asn-X-Ser or Asn-X-Thr in the protein amino acid sequence indicate potential glycosylation sites, with the exception that X cannot be His or Pro. Accordingly, various embodiments of the present invention provide CDKL5 polypeptides that have one or more asparagine (aka Asn or N) residues substituted with a different amino acid such as glutamine (aka Gln or Q) residues. One potential advantage of choosing glutamine for the substitution is that this amino acid is structurally similar to asparagine, with only an additional methylene unit present in the glutamine residue. However, other amino acids can also be used as substitutions for the asparagine residue(s). Alternatively, the glycosylation site can be altered by changing the third amino acid in the Asn-X-Ser or Asn-X-Thr sequence to another amino acid that is not serine (aka S or Ser) or threonine (aka T or Thr) and/or changing the second amino acid to histidine (aka H or His) or proline (aka P or Pro).
Embodiments of the present invention also provide CDKL5 polynucleotides that encode CDKL5 polypeptides that have one or more Asn residues substituted with another amino acid such as Gln residues. For example, one or more AAC, AAT or AAU sequences (which encode Asn) can be substituted with one or more CAA or CAG sequences (which encode Gln). Again, other alterations in the CDKL5 polynucleotides can encode other changes to the glycosylation sites such as substituting the second amino acid with His or Pro and/or changing the third amino acid to be another amino acid that is not Ser or Thr.
In one or more embodiments, the CDKL5 polypeptide has at least 98%, at least 98.5%, at least 99% or at least 99.5% sequence identity to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25. The CDKL5 polypeptide may contain deletions, substitutions and/or insertions relative to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more deletions, substitutions and/or insertions to the amino acid sequence described by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25.
In one or more embodiments, the CDKL5 polypeptide comprises one or more affinity-tags. In one or more embodiments, the affinity-tag is located on one or more of the N-terminus or the C-terminus of the CDKL5 polypeptide. Examples of tags that can be added to the fusion proteins include, but are not limited to, epitope tags (e.g. MYC, HA, V5, NE, StrepII, Twin-Strep-tag®, HPC4), glutathione S-transferase (GST), maltose-binding protein (MBP), calmodulin-binding peptide (CBP), FLAG®, 3×FLAG®, polyhistidine (His), and combinations thereof.
In one or more embodiments, the CDKL5 polypeptide comprises one or more protease cleavage sites. In some embodiments, the protease cleavage site is located on one or more of the N-terminus or the C-terminus of the CDKL5 polypeptide. Exemplary protease cleavage sites include, but are not limited to, cleavage sites sensitive to thrombin, furin, factor Xa, metalloproteases, enterokinases, cathepsin, HRV3C, TEV, and combinations thereof.
A variety of viral and cellular proteins possess basic polypeptide sequences that mediate translocation across cellular membranes. The capacity to translocate across cellular membranes has become an important tool for the delivery of high molecular weight polypeptides across membranes. The phrase “protein transduction domain” (PTD) and “cell-penetrating peptides” (CPPs) are usually used to refer to short peptides (<30 amino acids) that can traverse the plasma membrane of many, if not all, mammalian cells. After studies to identify the specific properties of the domain that allow them to collectively cross the plasma membrane, researchers have observed that these domains contain a large number of basic amino acid residues such as lysine and arginine. Thus, cell-penetrating peptides fall into two classes: the first consisting of amphipathic helical peptides that contain lysine residues which contribute a positive charge, while the second class includes arginine-rich peptides. These peptides could have therapeutic potential if used in combination with other proteins that are difficult to deliver to intracellular targets. The most frequent experimental uses of PTDs are TAT, Antennapedia (Antp), and other poly-arginine peptides.
Thus far, TAT has been the best characterized of the PTDs, and has been used to successfully deliver small cargoes, such as short peptides and oligonucleotides, to intercellular targets. HIV-TAT (HIV Transactivator of Transcription) is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1), and many studies have shown that TAT is able to translocate through the plasma membrane and reach the nucleus in order to activate transcription of the viral genome. Studies have also shown that TAT retains its penetration properties when coupled to several different proteins. In an effort to understand which areas of the TAT protein are critical to the translocation property, experiments have been conducted in which different length peptide fragments of TAT are synthesized and their penetration capabilities are assessed. (Lebleu et al. “A Truncated HIV-1 TAT Protein Basic Domain Rapidly Translocates through the Plasma Membrane and Accumulates in the Cell Nucleus.” J. Biol. Chem. 1997, 272:16010-16017). A region of basic amino acids has been identified as the aspect of TAT that retains this penetration property, and experiments in which a TAT protein without this basic amino acid cluster is unable to penetrate the cellular plasma membrane. In some instances, the shorter sequence cell-penetrating peptide has been modified to prevent cleavage during secretion by endoprotease enzymes such as furin. These modifications change the shortened cell-penetrating TAT amino acid sequence from YGRKKRRQRRR to YARKAARQARA, and this short peptide is referred to as TATκ.
The exact mechanism in which TAT is able to translocate across the plasma membrane remains uncertain. Recent work has explored the possibility that a special type of endocytosis is involved with TAT uptake, and a few cell lines have been identified that appear resistant to TAT penetration. The specific cargo to be delivered by TAT may also play a role in the efficacy of delivery. Previous research data have suggested that a TAT fusion protein has better cellular uptake when it is prepared in denaturing conditions, because correctly folded protein cargo likely requires much more energy (delta-G) to cross the plasma membrane due to structural constraints.
The capacity of the intracellular protein chaperones to refold the TAT cargo likely varies based on the identity and size of the protein cargo to be re-folded. In some instances, TAT-fusion proteins precipitate when placed in an aqueous environment and therefore cannot be prepared in a denatured manner nor remain stable for very long in native conformations. The design of the TAT-fusion protein must also be tailored to the specific cargo to be delivered. If the cargo protein is tightly associated at the N-terminus and the TAT domain is also found at the N-terminus, the TAT translocation domain may be buried in the cargo protein and transduction may be poor.
Numerous TAT-cargo variants have been successfully delivered into a variety of cell types, including primary culture cells, transformed cells, and cells present in mouse tissue. In culture, the TAT-fusion proteins generally diffuse easily into and out of cells, leading to a very rapid establishment of uniform concentration.
Many pharmaceutical agents such as enzymes, antibodies, other proteins, or even drug-loaded carrier particles need to be delivered intracellularly to exert their therapeutic action inside the cytoplasm, nucleus, or other specific organelles. Thus, the delivery of these different types of large molecules represents a significant challenge in the development of biologics. Current data suggest that TAT is able to cross the plasma membrane through more than one mechanism.
A TAT transduction domain has also been fused to the enzyme superoxide dismutase (SOD). (Torchilin, “Intracellular delivery of protein and peptide therapeutics.” Protein Therapeutics. 2008. 5(2-3):e95-e103). This fusion protein was used to demonstrate that it could translocate across cell membranes in order to deliver the SOD enzyme to the intracellular environment, and thus here the fusion protein has therapeutic potential in treating enzyme deficiency disorders that lead to higher accumulation of reactive oxygen species and oxidative stress on a host cell.
TAT fusion proteins have also been shown to transduce across the blood-brain barrier. A TAT domain fused to the neuroprotectant protein Bc1-xL was able to penetrate cells rapidly in culture, and when administered to mice suffering from cerebral ischemia, the fusion protein transduced brain cells within 1-2 hours. After transduction, the cerebral infarct was reduced in size in a dose-dependent manner (Cao, G. et al., “In Vivo Delivery of a Bc1-xL Fusion Protein Containing the TAT Protein Transduction Domain Protects against Ischemic Brain Injury and Neuronal Apoptosis.” J. Neurosci. 22, 5423, 2002.)
In various embodiments, the CDKL5 variants described herein are operably linked to a CPP such as TAT, modified TAT (TATκ), Transportan, Antennapedia or P97. As used herein, TAT can refer to the original TAT peptide having 11 amino acids (designated TAT11) or can refer to a TAT peptide having an additional 16 N-terminal amino acids (designated as TAT28) that are derived from the polylinker of the plasmid used for cloning. Similarly, TATκ can refer to a modified version of TAT11 (designated TATκ11) or a modified version of TAT28 (designated TATκ28). The TATκ28 can be further modified (designated TATκκ28) to remove a potential additional weak furin site. The amino acid sequences of the CPPs TAT28, TATκ28, TAT11, TATκ11, Transportan, Antennapedia, P97 and TATκκ28 are provided in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 167, respectively.
In some embodiments, the CPP has at least 90% sequence identity to SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 167. In some embodiments, the CPP has at least 95% sequence identity to SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 167. In some embodiments, the CPP has 100% sequence identity to SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 167. In some embodiments, the CPP has at least 90% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 167. In some embodiments, the CPP has at least 95% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 167. In some embodiments, the CPP has 100% sequence identity to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 167. In various embodiments, the CPP does not have the sequence of SEQ ID NO: 34.
In various embodiments, the CPP can have an N-terminal glycine added. For example, TATκ28 and TAT28 would otherwise have an N-terminal aspartate residue, which has a low stability. Adding an N-terminal glycine to the sequence can increase protein stability via the N-end rule. Accordingly, in some embodiments, any of the fusion proteins that have a leader signal polypeptide can have a glycine added at the C-terminal end of the leader signal polypeptide, such that upon cleavage of the leader signal polypeptide, the new N-terminus of the fusion protein will begin with glycine. In an analogous manner, those fusion proteins lacking a leader signal polypeptide can also have a glycine added between the N-terminal methionine and the remainder of the fusion protein. Also in analogous manner, those fusion proteins having a CPP other than TAT28 or TATκ28, can also have a glycine added between a leader signal polypeptide and a CPP.
In one or more embodiments, the CPP is operatively coupled to the N-terminus of the CDKL5 polypeptide. In one or more embodiments, the CPP is operatively coupled to the C-terminus of the CDKL5 polypeptide.
In one or more embodiments, the CPP comprises one or more affinity-tags. In one or more embodiments, the affinity-tag is located on one or more of the N-terminus or the C-terminus of the CPP. Examples of affinity-tags that can be added to the CPP include, but are not limited to, epitope tags (e.g. MYC, HA, V5, NE, StrepII, Twin-Strep-tag®, HPC4), glutathione S-transferase (GST), maltose-binding protein (MBP), calmodulin-binding peptide (CBP), FLAG®, 3×FLAG® polyhistidine (His), and combinations thereof.
In one or more embodiments, the CPP comprises one or more protease cleavage sites. In some embodiments, the protease cleavage site is located on one or more of the N-terminus or the C-terminus of the CPP. Exemplary protease cleavage sites include, but are not limited to, cleavage sites sensitive to thrombin, furin, factor Xa, metalloproteases, enterokinases, cathepsin, HRV3C, TEV, and combinations thereof.
As described above, CDKL5 variants can be used in fusion proteins, such as proteins that also contain a CPP. Other polypeptides can also be incorporated into such fusion proteins, such as leader signal polypeptides to enhance protein secretion or affinity-tags for detecting and/or purifying the fusion proteins, as well as linker polypeptides that can be used to link functional polypeptides.
Examples of leader signal polypeptides include, but are not limited to, modified fragments of human immunoglobulin heavy chain binding protein (modified BiP, e.g. SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 168), murine Igκ chain leader polypeptide (SEQ ID NO: 42, e.g. pSecTag2 from ThermoFisher vectors) or insulin growth factor peptides (IGF2) such as the wild-type IFG2 (SEQ ID NO: 156) or variants thereof (e.g. SEQ ID NOS 157-166). Examples of modified BiP signal polypeptides include those described in U.S. Pat. No. 9,279,007, which is hereby incorporated by reference in its entirety. Other examples of modified BiP signal polypeptides include mvBIP, which has a valine added before the lysine in mBiP as shown in SEQ ID NO: 168.
In one or more embodiments, the fusion protein comprises a CDKL5 polypeptide having an N-terminal CPP, optionally with a leader signal polypeptide before the N-terminal CPP. In one or more embodiments, the fusion protein comprises a CDKL5 polypeptide having a C-terminal CPP, optionally with a leader signal polypeptide before the CDKL5 polypeptide. In one or more embodiments, the fusion protein comprises a leader signal peptide and a CDKL5 polypeptide without a CPP.
Examples of affinity-tags that can be added to the fusion proteins include, but are not limited to, epitope tags (e.g. MYC, HA, V5, NE, StrepII, Twin-Strep-tag®, HPC4), glutathione S-transferase (GST), maltose-binding protein (MBP), calmodulin-binding peptide (CBP), FLAG®, 3×FLAG®, polyhistidine (His), and combinations thereof.
Some embodiments of the fusion protein may also include a protease cleavage site. In some embodiments, the protease cleavage site is located on the N-terminus of affinity-tag. In some embodiments, the protease cleavage site is located on the C-terminus of affinity-tag. Exemplary protease cleavage sites include, but are not limited to, cleavage sites sensitive to thrombin, furin, factor Xa, metalloproteases, enterokinases, cathepsin, HRV3C, TEV and combination thereof.
The recombinant protein (e.g. CDKL5 variant or fusion protein) can be expressed in and secreted from host cells using appropriate vectors. For example, mammalian cells (e.g., CHO, HeLa or HEK cells), insect cells (e.g. Sf9 or BTI-Tn-5B1-4) or bacterial cells (e.g., E. coli or P. haloplanktis TAC 125 cells) can be used. Exemplary plasmids are described in the examples below and shown in
After expression and secretion, recombinant protein can be recovered and purified from the surrounding cell culture media using standard techniques. Alternatively, recombinant protein can be isolated and purified directly from cells, rather than the medium.
In some embodiments, the BTI-Tn-5B1-4 cells are used to express and purify CDKL5 variant or fusion protein.
For lysis, the cells expressing the CDKL variant or fusion protein may be pelleted and subsequently resuspended into a lysis buffer. The resuspended cells may be then incubated in a cavitation chamber that is charged from about 100 PSI to about 2000 PSI with nitrogen gas. The resuspended cells may be incubated in the charged cavitation chamber for about 5 minutes to about 60 minutes. In some embodiments, the resuspended cells may be incubated in the cavitation chamber charged to 750 PSI with nitrogen gas. In some embodiments, the resuspended cells may be incubated in the charged cavitation chamber for 15 minutes. An effluent from the cavitation chamber after incubation may be then transferred on ice. A detergent may be added in the effluent followed by incubation on ice for about 5 minutes to about 60 minutes. In some embodiments, the detergent is added in the amount of about 0.1% (w/v) to about 5% (w/v). In some embodiments, the detergent is Triton X-100. The effluent with the detergent is then sonicated to lyse the cells. After lysis, soluble fractions and insoluble fractions may be separated. In some embodiments, the soluble fraction and insoluble fraction may be separated by centrifugation. The soluble material may be filtered. In some embodiments, the soluble material may be filtered through 0.45 μm filter.
For purification of the CDKL5 variants or the fusion protein, the filtered soluble material is then subject to purification. In some embodiments, the CDKL5 variants or the fusion protein is purified by a chromatography technique. In some embodiments, the chromatography technique is an affinity chromatography. In some embodiments, the CDKL5 variant or the fusion protein comprises one or more affinity tags. In some embodiments, the affinity-tag include, but are not limited to, epitope tags (e.g. MYC, HA, V5, NE, StrepII, Twin-Strep-tag®, HPC4), glutathione S-transferase (GST), maltose-binding protein (MBP), calmodulin-binding peptide (CBP), FLAG®, 3×FLAG®, polyhistidine (His) and combination thereof. In some embodiments, the CDKL5 variant or the fusion protein has a Twin-Strep-tag®. In some embodiments, the CDKL5 variant or the fusion protein with the affinity-tag is purified on a purification resin. In some embodiments of the CDKL5 variant or the fusion protein with a Twin-Strep-tag®, the purification resin is a strep-tactin resin.
Some embodiments of the CDKL5 variant or the fusion protein may also include one or more protease cleavage sites. In some embodiments, the protease cleavage site is located on the N-terminus of the CDKL5 variant or the fusion protein. In some embodiments, the protease cleavage site is located on the C-terminus of the CDKL5 variant or the fusion protein. In some embodiments, the protease cleavage site is located on N-terminus and C-terminus of the CDKL5 variant or the fusion protein. In some embodiments, the cleavage is performed when the CDKL5 variant or the fusion protein is bound to the purification resin. In some embodiments, the cleavage is performed when the CDKL5 variant or the fusion protein with the Twin-Strep-tag® is bound to the strep-tactin resin.
In one or more embodiments, a subject may be administered with the CDKL5 protein or variants or fusion proteins. In some embodiments, the subjects may be humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys etc. In some embodiments, the subject is a human.
In one or more embodiments, a cellular uptake of the CDKL5 protein or variants or fusion proteins is determined in cells isolated from the subject. In some embodiments, the cells may be isolated from rats. In some embodiments, the cells may be neuronal cells. In some embodiments, the cells may be embryonic primary cortical neurons. In some embodiments, the embryonic primary cortical neurons may be isolated from rats. In some embodiments, the cells may be cultured and incubated with the CDKL5 protein or variants for a duration of time. The duration of time may be at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes or at least 60 minutes. In some embodiments the duration of time may be from 5 minutes to 24 hours, 15 minutes to 24 hour, 30 minutes to 24 hour, 1 hour to 24 hour, 4 hour to 24 hour, 8 hour to 24 hour, 12 hour to 24 hour, 5 minutes to 12 hours, 15 minutes to 12 hour, 30 minutes to 12 hour, 1 hour to 12 hour, 2 hour to 12 hour, 4 hour to 12 hour, 6 hour to 12 hour, 8 hour to 12 hour, 10 hour to 12 hour, 5 minutes to 6 hours, 15 minutes to 6 hour, 30 minutes to 6 hour, 1 hour to 6 hour, 1.5 hour to 6 hour, 2 hour to 6 hour, 2.5 hour to 6 hour, 3 hour to 6 hour, 4 hour to 6 hour 5 hour to 6 hour, 5 minutes to 4 hours, 15 minutes to 4 hour, 30 minutes to 4 hour, 1 hour to 4 hour, 1.5 hour to 4 hour, 2 hour to 4 hour, 2.5 hour to 4 hour, 3 hour to 4 hour, 5 minutes to 2 hours, 15 minutes to 2 hour, 30 minutes to 2 hour, 1 hour to 2 hour, 1.5 hour to 2 hour, 5 minutes to 1 hours, 15 minutes to 1 hour or 30 minutes to 1 hour.
Any of the CDKL5 polypeptides and/or fusion proteins described herein can be utilized in gene therapy via an appropriate polynucleotide (e.g. DNA or RNA) encoding the desired CDKL5 polypeptide and/or fusion protein.
In various embodiments, gene therapy is provided through the use of a composition comprising a gene therapy delivery system and a CDKL5 polynucleotide. Exemplary gene therapy delivery systems include, but are not limited to, viral vectors, liposomes, lipid-nucleic acid nanoparticles, exosomes and gene editing systems. For example, a gene editing system such as Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) associated protein 9 (CRISPR-Cas-9), Transcription activator-like effector nucleases (TALEN) or ZNF (Zinc finger proteins) can be used to insert the CDKL5 polynucleotide into the DNA of the host cell.
Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, lentiviral vectors, retroviral vectors, poxviral vectors or herpes simplex viral vectors. Viral vectors typically utilize a viral particle (virion) including an outer protein shell (capsid) and one or more DNA or RNA sequences (viral polynucleotides) encapsulated in the capsid. For example, AAV vectors typically include one or more inverted terminal repeat (ITR) sequences, a replication (Rep) gene sequence, and a capsid (Cap) gene sequence. The ITR, Rep and Cap sequences may be included in the same plasmid (in cis), or may be provided in separate plasmids (in trans). The capsid may be derived from the same serotype as the ITR sequences, or the AAV vector can be a hybrid vector utilizing ITR sequences and capsids derived from different AAV serotypes. Exemplary AAV serotypes include AAV1, AAV 2, AAV 3, AAV 4, AAV 5, AAV 6, AAV 7, AAV 8, AAV 9, AAV10, AAV11, hybrid serotypes, and synthetic serotypes. An exemplary set of ITRs is provided in SEQ ID NO: 27 (L-ITR) and SEQ ID NO: 28 (R-ITR), which are derived from AAV2.
The viral vectors also may include additional elements for increasing expression and/or stabilizing the vector such as promoters (e.g., hybrid CBA promoter (CBh) and human synapsin 1 promoter (hSyn1)), a polyadenylation signals (e.g. Bovine growth hormone polyadenylation signal (bGHpolyA)), stabilizing elements (e.g. Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE)) and/or an SV40 intron. The DNA sequences for CBh and hSyn1 are provided in SEQ ID NO: 29 and SEQ ID NO: 30, respectively.
The gene therapy delivery system can be utilized to deliver the CDKL5 polynucleotide to the target cells so that the CDKL5 polypeptide (or fusion protein comprising the same) can be expressed in the target cells. In various embodiments, the CDKL5 polypeptide (e.g. wild-type CDKL5 polypeptides, CDKL5 variants with one or more N-linked glycosylation sites removed and/or shorter CDKL5 variants) (or fusion protein comprising the same) is expressed in the target cell and utilized in the same cell. In other embodiments, the CDKL5 polypeptide (or fusion protein comprising the same) is expressed in a first cell, secreted, and then penetrates into a second cell. In such embodiments, a leader signal polypeptide and/or a cell-penetration may be used to enhance secretion and/or penetration of the CDKL5 polypeptide. Without wishing to be bound by any particular theory, it is believed that secretion and penetration of CDKL5 polypeptide can be used to enhance the effects of gene therapy over conventional gene therapy approaches that only introduce DNA and RNA into the patient, as transduction in gene therapy may only be limited to a certain portion of the patient's cells (e.g. 10% of the target patient cells are successfully transduced with the DNA/RNA). In this way, the successfully transduced cells may be used to express the CDKL5 polypeptide (or fusion protein comprising the same) for both the transduced cells and neighboring cells that were not successfully transduced.
Another aspect of the invention can include cross-correction. The genetherapy may not be effective to successfully transfect all defective cells. In one or more embodiments, a genetic defect in non-transfected cells can be corrected by the neighboring successfully transfected cells. For example, the CDKL5 polypeptide or fusion protein may be expressed in a successfully transfected cell, secreted from that cell, and taken up by a neighboring cell that was not successfully transfected. The defect may be cross-corrected by any of the gene therapy methods described herein via an appropriate polynucleotide (e.g. DNA or RNA) encoding the desired CDKL5 polypeptide and/or fusion protein. Any of the CDKL5 polypeptides and/or fusion proteins described herein can be utilized to cross-correct a CDKL5-related defect.
In one or more embodiments, a CDKL5 null subject is used for determining the fusion protein induced cross-correction. In some embodiments, the subject is a mouse. In some embodiments, a viral vector may be used to correct the CDKL5 defect. In a particular embodiment, AAV vector was used to correct the CDKL5 defect. In a particular embodiment, the AAV vector comprises a AAV-PHP.B.CBH.BIP-TATκ28-CDKL5.SV40. In a particular embodiment, the viral vector comprising corrective gene is administered in a dose sufficient to correct the genetic defect. In some embodiments, the sufficient dose for correcting genetic defect in mice is in a range of 10×e2 GC/mice to 10×e15 GC/mice. In some embodiments, the sufficient dose for correcting genetic defect in mice may be 10×e2 GC/mice, 10×e3 GC/mice, 10×e4 GC/mice, 10×e5 GC/mice, 10×e6 GC/mice, 10×e7 GC/mice, 10×e8 GC/mice, 10×e9 GC/mice, 10×e10 GC/mice, 10×e11 GC/mice, 10×e12 GC/mice, 10×e13 GC/mice, 10×e14 GC/mice or 10×e15 GC/mice. Exemplary routes of administration include, but are not limited to, intrathecal, intravenous, intracisternal, retro-orbital, intraperitoneal, intracerebroventrical or intraparenchymal administration.
In one or more embodiments, the CDKL5 null mice may be divided into a treatment group and a control group. Each group, the treatment group and the control group, may further be divided into two subgroups based on route of administration. More than one route can be used concurrently, if desired. In one or more embodiments, each subgroup may be administered AAV-PHP.B.CBH.BIP-TATκ28-CDKL5.SV40 dose through either intracerebroventricular (ICV) or retro orbital (RO) route of administration. Each subgroup received AAV-PHP.B.CBH.BIP-TATκ28-CDKL5.SV40 dose in an amount of 10×e8 GC/mice, 10×e9 GC/mice or 10×e10 GC/mice. Three months post-administration, the impact of the vector on behavioral endpoints may be assessed and the mice may be euthanized for transgene expression analysis.
After euthanizing mice, various section of brain may be taken including but not limited to sagittal section. The sections may be immunostained with DAPI, anti-NeuN antibody, anti-CDKL5 RNA antibody and anti-CDKL5 protein antibody. The sections may be taken from isocortex, striatum, thalamus and hippocampal formation section of brains.
The immunostained images may be analyzed using Visiopharm software. The immunostained cells may be divided into six groups: (1) DAPI stain to identify cells; (2) NeuN stain to identify neurons; (3) Neurons having CDKL5 mRNA and CDKL5 protein; (4) Neurons having CDKL5 mRNA; (5) Cross-corrected neurons; and (6) Cross-corrected non-neurons. The result of image analysis may be further subject to a statistical analysis for cross-corrected neurons and non-neurons.
The gene therapy compositions (e.g. comprising CDKL5 polynucleotides) or the protein replacement therapy compositions (e.g. comprising recombinant proteins including CDKL5 variants or fusion proteins), can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings. For example, in one or more embodiments, a composition for intravenous administration is a solution in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
Gene therapy compositions (e.g. comprising CDKL5 polynucleotides) or protein replacement therapy compositions (e.g. comprising recombinant proteins including CDKL5 variants or fusion proteins) (or a composition or medicament containing the gene therapy composition or protein replacement therapy composition) are administered by an appropriate route. In one or more embodiments, the gene therapy composition or protein replacement therapy composition is administered intravenously. In other embodiments, the gene therapy composition or protein replacement therapy composition is administered by direct administration to a target tissue, such as to heart or skeletal muscle (e.g., intramuscular; intraventricularly), or nervous system (e.g., intrathecal delivery—delivery into the space under the arachnoid membrane of the brain or spinal cord). More than one route can be used concurrently, if desired. Exemplary routes of administration include, but are not limited to, intrathecal, intravenous, intracisternal, intracerebroventrical or intraparenchymal administration.
The gene therapy composition (e.g. comprising CDKL5 polynucleotides) or protein replacement therapy composition (e.g. comprising recombinant protein including CDKL5 variants or fusion proteins) (or a composition or medicament containing such gene therapy composition or protein replacement therapy) is administered in a therapeutically effective amount (e.g., a dosage amount that, when administered at regular intervals, is sufficient to treat the disease, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or lessening the severity or frequency of symptoms of the disease). The amount which will be therapeutically effective in the treatment of the disease will depend on the nature and extent of the disease's effects. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed will also depend on the route of administration, and the seriousness of the disease, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
The therapeutically effective amount of gene therapy composition (e.g. comprising CDKL5 polynucleotides) or protein replacement therapy composition (e.g. comprising recombinant protein including CDKL5 variants or fusion proteins) (or a composition or medicament containing such gene therapy composition or protein replacement therapy) can be administered at regular intervals, depending on the nature and extent of the disease's effects, and/or on an ongoing basis. Administration at a “regular interval,” as used herein, indicates that the therapeutically effective amount is administered periodically (as distinguished from a one-time dose). The administration interval for a single individual need not be a fixed interval, but can be varied over time, depending on the needs of the individual.
The gene therapy composition (e.g. comprising CDKL5 polynucleotides) or protein replacement therapy composition (e.g. comprising recombinant protein including CDKL5 variants or fusion proteins) (or a composition or medicament containing such gene therapy composition or protein replacement therapy composition) may be prepared for later use, such as in a unit dose vial or syringe, or in a bottle or bag for intravenous administration. Kits containing the gene therapy composition (e.g. comprising CDKL5 polynucleotides) or protein replacement therapy composition (e.g. comprising recombinant protein including CDKL5 variants or fusion proteins) (or a composition or medicament containing such gene therapy composition or protein replacement therapy composition), as well as optional excipients or other active ingredients, such as other drugs, may be enclosed in packaging material and accompanied by instructions for reconstitution, dilution or dosing for treating a subject in need of treatment, such as a patient having a CDKL5 deficiency, Rett syndrome, or a Rett syndrome variant.
In those constructs wherein CDKL5 is fused C-terminally to additional N-terminal amino acid sequences, the initial methionine (amino acid 1) of CDKL5 is removed. In these constructs, the CDKL5 polypeptide begins with the second amino acid, lysine. Although specific reference is made to N-terminal amino acid sequences (e.g. N-terminal CPPs), C-terminal amino acid sequences (e.g. C-terminal CPPs) are also encompassed by the present disclosure.
The abbreviations used in
Mus musculus (mouse) DHFR allows auxotrophic selection of
E. coli cells
SEQ ID NO: 84 in insect cells. This fusion protein comprises TATκ11 and the full-length human CDKL5107 isoform without a leader signal polypeptide.
Various CDKL5 fusion proteins were expressed in E. coli, CHO, HEK and insect cells, as well as using in vitro transcription/translation with HeLa cell lysates, as further described below.
Full-length and truncations of TATκ28-CDKL5_107-FH were cloned into the pET vector, pEX-1, and transformed into the E. coli strain, BL21(DE3). Colony-purified transformants were cultured in LB+100 μg/mL ampicillin at 37° C. to exponential phase. The cultures were then cooled to 20° C. and induced with (or without) 1 mM IPTG for 16 hours. Cell pellets were collected and lysed in B-Per Complete Bacterial Protein Extraction Solution (Thermo) supplemented with 1X Complete Protease Inhibitor Complex (Roche). Lysis was allowed to proceed for 30 minutes at room temperature. A soluble fraction was prepared from the lysate by centrifugation at 16,000×g for 15 minutes at 4° C. Proteins were resolved on SDS-PAGE, transferred to nitrocellulose membranes, probed with a rabbit anti-polyhistidine antibody (Thermo), and detected with a fluorescent secondary antibody.
Blots shown
For
For
CHO-S cells (20×10{circumflex over ( )}6cells) were electroporated using Maxcyte STX with 8 plasmids: (1) pOptiVec empty vector; 2) TATκ28-CDKL5-107-3×FlagHis; 3) TATκ11-CDKL5-107-3×FlagHis; 4) TAT11-CDKL5-107-3×FlagHis; 5) TAT28-CDKL5-107-3×FlagHis; 6) ANTP-CDKL5-107-3×FlagHis; 7) TRANSP-CDKL5-107-3×FlagHis and 8) MBiP-TATκ28-CDKL5-107-3×FlagHis (coding sequences being CHO codon-optimized). Cells were recovered in culture medium, and cultured for one day. Cells were harvested and lysed. For each transfection, 20 μg lysate was subjected to 4-12% BisTris SDS-PAGE, and transferred to nitrocellulose blot using the iBlot2 system. The blot was blocked in 5% milk in 1×TBS-T. Blot was subjected to Western blot by incubating with 1:2000 dilution of rabbit anti-His antibody overnight. After a series of washes, blot was incubated with 1:10000 anti-rabbit IgG DyaLight 680 secondary antibody. Additional washes were performed. Blot was imaged on Licor Odyssey scanner. Blot shown in
HEK293F cells (8×10{circumflex over ( )}cells) were transfected with FuGeneHD (240 FuGeneHD: 8 μg DNA ratio) and 7 plasmids: 1) empty pOptiVec; 2) TATκ11-CDKL5_107-3×FlagHis; 3) TAT11-CDKL5_107-3×FlagHis; 4) TAT28-CDKL5_107-3×FlagHis; 5) ANTP-CDKL5_107-3×FlagHis; 6) TRANSP-CDKL5_107-3×FlagHis and 7) TATκ28-CDKL5_107-3×FlagHis (coding sequences being human codon-optimized). Cells were incubated and harvested 2 days post transfection. Cells were lysed, and 20 μg lysate was subjected to 4-12% BisTris SDS-PAGE, and transferred to nitrocellulose blot using the iBlot2 system. The blot was blocked in 5% milk in 1×TBS-T. Blot was subjected to Western blot by incubating with 1:2000 dilution of rabbit anti-His antibody overnight. After a series of washes, blot was incubated with 1:10000 anti-rabbit IgG DyaLight 680 secondary antibody. Additional washes were performed. Blot was imaged on Licor Odyssey scanner. Blot shown in
Methotrexate amplification was used to amplify expression of CDKL5 fusion proteins in CHO-DG44 cells. TATκ28-CDKL5_107-FH (no signal sequence), Igκ-TATκ28-CDKL5_107-FH, and mBiP-TATκ28-CDKL5_107-FH were cloned into the pOptiVec vector providing the DHFR gene for methotrexate resistance. These plasmids were transfected into DG44 cells (deficient in dhfr) and selected by growth in medium deficient in hypoxanthine and thymidine. Methotrexate-resistant subcultures were obtained by culturing the cells sequentially in 0.1, 0.25, 0.5, and 1 μM Methotrexate (MTX), allowing cells to recover to 70% viability between steps. Cell pellets were lysed in 50 mM Tris-HCl with 75 mM NaCl, 1% Triton X-100, and 1.5X protease inhibitor cocktail (EDTA-free), pH 7.4. 40 μg of total protein were resolved on LDS-PAGE, transferred to nitrocellulose membranes, probed with a rabbit anti-polyhistidine antibody (Thermo), and detected with a fluorescent secondary antibody.
The blot shown in
In addition to the DG44 transfected cell lines noted above (TATκ28-CDKL5_107-FH without a signal sequence, Igκ-TATκ28-CDKL5_107-FH, and mBiP-TATκ28-CDKL5_107-FH), an Igκ-TATκ28-eGFP-CDKL5_107-MH plasmid stably transfected in adherent HEK293T cells were compared for secretion of CDKL5 fusion protein into the culture medium and in the cell lysates. The mBiP-TATκ28-CDKL5_107-FH cell line was represented by both 0 mM MTX and 0.5 μM MTX sub-cultures. After two days in serum-free growth, the conditioned medium was collected and concentrated 200-fold.
Cell pellets were lysed in 50 mM Tris-HCl with 75 mM NaCl, 1% Triton X-100, and 1.5X protease inhibitor cocktail (EDTA-free), pH 7.4. Cell lysates or concentrated conditioned medium were resolved on LDS-PAGE, transferred to nitrocellulose membranes, probed with a rabbit anti-polyhistidine antibody (Thermo), and detected with a fluorescent secondary antibody.
Blots shown in
A single plasmid (pCHO 1.0) harboring both TATκ28-CDKL5-FH (no signal sequence) and one of several putative CDKL5 substrates (HOMER1, HDAC4, ARHGEF2, MAPRE2, AMPH1, or SHANK1), or no protein partner, were transiently transfected into HEK293F cells. After five days in culture, cells were harvested and lysed in 50 mM sodium phosphate, 150 mM sodium chloride, 0.5% Triton-X100, 1X Complete Protease Inhibitor Complex, EDTA-free, pH 7 for 30 minutes at 4° C. A soluble fraction was obtained by centrifugation of the lysates at 16,000×g for 15 minutes at 4° C. Soluble protein was determined by BCA assay and an equal quantity was resolved on SDS-PAGE, transferred to nitrocellulose membranes, probed with rabbit anti-polyhistidine (ThermoFisher) and mouse anti-CDKL5 antibodies (EMD Millipore), and detected with near-infrared fluorescent secondary antibodies, anti-rabbit IgG DyaLight 680 and anti-mouse IgG DyaLight 800 (Cell Signaling Technology). As shown in the blot of
The following proteins were cloned into a T7/EMCV-IRES plasmid (pT7CFE1): eGFP, CDKL5_115 and TAT28-CDKL5_107-FH. Purified plasmid DNA was introduced into a HeLa cell-based IVT kit (Thermo) for non-CAP dependent combined in vitro transcription/translation for 5 hours at 30° C. Protein samples were resolved on SDS-PAGE, transferred to nitrocellulose membranes, probed with a rabbit anti-polyhistidine (His) antibody (Thermo), and detected with a fluorescent secondary antibody. Blot shown in
Further analysis of MBiP-TATκ28-CDKL5-107-3×FlagHis revealed that this fusion protein was glycosylated when expressed in CHO-DG44 and HEK293F cells. Plasmids were transiently transfected by electroporation into CHO-DG44 and HEK293F cells. Cell pellets were lysed and a soluble fraction was obtained by centrifugation. The soluble fraction was denatured in PNGase F buffer and incubated with PNGase F to remove N-linked glycans. Digested samples were resolved by SDS-PAGE, transferred to nitrocellulose and immunoblotted with an anti-polyhistidine antibody. Blot shown in
Other expression systems were also investigated to improve expression, reduce glycosylation and/or enhance purification. One such system utilized the insect cells Sf9. To protect the N-terminus of TATκ28-CDKL5 and other CDKL5 fusion proteins, a GST tag was genetically fused to the N-terminus, separated from the remaining portion of the CDKL5 fusion protein by an HRV3C protease site. Another HRV3C protease site was added to the C-terminus of the CDKL5 protein to separate the FLAG and polyhistidine (His) affinity tags. Sf9 cells were co-transfected with linearized baculovirus (BV) DNA and transfer plasmids: 1) GST-P-TATκ28-eGFP-P-FH; 2) GST-P-eGFP-P-FH; 3) GST-P-TAT28-CDKL5_107-P-FH; 4) GST-P-TATκ28-CDKL5_107-P-FH; 5) GST-P-p97p-CDKL5_107-P-FH; 6) GST-P-Antp-CDKL5_107-P-FH; 7) GST-P-TAT11-CDKL5_107-P-FH and GST-P-Transp-CDKL5_107-P-FH (coding sequences being Sf9 codon-optimized). 1 μg protein run out on duplicate 4-12%, 10-well NuPage gels. Gels run at 175V for 90 minutes. Protein transferred to nitrocellulose using the iBLOT at 20v for 7 minutes. Expression of CDKL5 fusion proteins was analyzed with Sypro Ruby Red total protein stain as shown in
CDKL5 fusion proteins from insect cells were also purified to isolate the CDKL5 proteins from the cell lysate. GST-P-TATκ28-CDKL5_107-P-FH proteins were expressed in High Five (BTI-Tn-5B1-4) cells maintained as suspension cultures in Sf90011 media. Infected cell pellets were lysed with 50 mM NaPO4, 500 mM NaCl, 10% Glycerol, pH 6) supplemented with 1X HALT Protease Inhibitor cocktail without EDTA (Thermo, 78437), 1 mM tris 2-carboxyethyl-phosphine (TCEP) and 5 mM EDTA at a ratio of 10 ml Lysis Buffer per 100 million cells. Following lysis by nitrogen cavitation using the Parr 4639 Cell Cracker at 750PSI for 15 minutes, Triton X-100 was added to 0.5%. The lysate was clarified by centrifugation at 31,000×g for 20 minutes. The soluble material was adjusted to 350 mM NaCl and applied to HiTrap SP Fast Flow resin (GE Healthcare, 17-5157-01). Bound protein was eluted with a 10 column volume (CV) NaCl gradient, 350-2000 mM. The CDKL5 protein peak, 525-1225 mM NaCl, was buffer-exchanged in to Buffer B (50 mM NaPO4, 500 mM NaCl, 10% Glycerol, 1X HALT Protease inhibitor cocktail without EDTA, 1 mM TCEP, pH 8). Protein was applied to IMAC Sepharose 6 FF resin (GE Healthcare, 17-0921-09) that had been charged with Nickel Sulfate and pre-equilibrated with Buffer B. The resin was washed with Buffer B+60 mM imidazole. The resin with incubated with 40 U of HRV3C protease (Millipore, 71493) at 4° C. up to overnight to remove the GST, FLAG and polyhistidine (His) affinity tags. Aliquots of the cleaved material examined at 3 hours and overnight. The resin was washed with 50 mM NaPO4, 500 mM NaCl, 10% Glycerol, 1 mM TCEP+1X HALT PI-EDTA+0.5% Triton X-100+500 mM imidazole to elute the CDKL5. The eluted protein lacks the affinity tags and migrates more quickly though SDS-PAGE.
GST-P-TATκ28-CDKL5_107-P-FH expressed in HighFive cells via infection with baculovirus was released from cells by lysis in 50 mM Na-phosphate, 500 mM NaCl, 10% glycerol, 1 mM TCEP, 1 mM EDTA, 1×HALT protease inhibitor cocktail, pH 6.0, using nitrogen cavitation for 15 minutes at room temperature. Following cell disruption, Triton X-100 was added to 0.5%, and incubated for 30 minutes at 4° C. The lysate was separated into soluble and insoluble fractions by centrifugation at 15,000×g for 15 minutes at room temperature. The soluble fraction was then further modified with the following conditions by dilution to the same final volume:
Following incubation for 1 hour at room temperature under the described conditions, the solutions were again separated into soluble and insoluble fractions by centrifugation. The insoluble fraction was re-suspended in a volume equal to the soluble fraction, and both soluble and insoluble fractions were resolved on LDS-PAGE, then detected by staining with Coomassie.
In this Example, the fusion protein TwinStrep-HRV3C-TATκ28-CDKL5-HRV3C-FLAG-His-HPC4 was expressed and purified.
CDKL5 fusion proteins from insect cells were purified to isolate the CDKL5 proteins from the cell lysate.
In this Example, the fusion protein was TwinStrep-HRV3C-TATκ28-CDKL5-HRV3C-FLAG-His-TwinStrep protein. The fusion protein has an amino acid sequence according the SEQ ID No: 176. Similarly, the fusion protein has a nucleotide sequence according SEQ ID No: 177.
For lysis, the cell pellet was resuspended in a lysis buffer (50 mM Tris HCl, 500 mM NaCl, 10% Glycerol, 1 mM EDTA at pH 8) supplemented with 1X HALT Protease Inhibitor cocktail without EDTA (Thermo, 78437). Following lysis by nitrogen cavitation using the Parr 4639 Cell Cracker at 750PSI for 15 minutes, Triton X-100 was added to 0.5%. The lysate was clarified by centrifugation at 31,000×g for 20 minutes. The clarified lysate was collected into a soluble fraction.
The insoluble pellet was washed with the lysis buffer. The washed insoluble pellet was then resuspended in 2 ml of the lysis buffer and sonicated. The soluble fraction after sonication was used for protein analysis. BCA assay was used to measure the protein concentration. NuPAGE was used to analyze the protein expression in insect cells. In
For purifying the fusion protein from other soluble proteins, Strep-Tectin resin was used. The soluble fraction was loaded on a pre-equilibrated Strep-Tectin column. The affinity-tags were cleaved off on Strep-Tectin column using His-HRV3C protease. For the cleavage, the fusion protein bound to Strep-Tectin was incubated with the His-HRV3C protease for about 1 hour. After the digestion, the flow through and wash were collected. In
For a buffer exchange of the digested fusion protein and the His-HRV3C protease, HiPrep 26/20 Desalting column (Cytiva 17-5087-01) was used. The column was pre-equilibrated with Buffer A (50 mM Bis-Tris, 350 mM NaCl, 10% (v/v) glycerol at pH 6). The fusion protein containing the His-HRV3C protease was loaded on the column and fractions were pooled together into a desalting pool.
For purifying the fusion protein from the His-HRV3C protease, SP Sepharose capture column was used. The desalting pool was applied to an SP Sepharose capture, which was pre-equilibrated with the Buffer A. The TATκ28-CDKL5 protein was eluted with 55% of Buffer A and 45% of Buffer B (50 mM Bis-Tris, 2000 mM NaCl, 10% (v/v) glycerol at pH 6) to remove His-HVRc3 from the purified TATκ28-CDKL5 protein fraction.
In this Example, an uptake of CDKL5 fusion proteins in embryonic primary cortical neurons was determined. The embryonic primary cortical neurons were isolated from healthy rat embryos at E15. The embryonic primary cortical neurons were seeded on poly-1-lysine coated glass coverslips and maintained for 14 days in vitro (DIV14). Recombinant TATκ28-CDKL5 was purified from a baculoviral/insect cell expression system via affinity-tag chromatography. The affinity-tags were removed by protease cleavage and the full-length protein was further isolated and concentrated via cation exchange chromatography. Cultured embryonic primary cortical neurons were treated with 10 μg/ml recombinant TATκ28-CDKL5 for 6 hours. Non-treated cultured embryonic primary cortical neurons were used as a negative control. Each sample, either treated or non-treated, were fixed in 4% PFA, permeabilized in 0.1% saponin, and stained using anti-MAP2, anti-CDKL5, and/or anti-phosphorylated (S222) EB2 antibodies. The cells were counterstained with DAPI and mounted on glass microscope slides under Prolong Diamond anti-fade mounting medium. The samples were imaged using a Leica SP8 point scanning laser confocal microscope with a 63x oil-immersion objective. The images were processed using Leica Lightning software and merged and colorized using ImageJ software. Analysis of phospho (S222) EB2 signal was performed using ImageJ software and graphed with GraphPad Prism software.
Similar experiments were also performed in rat DIV7 embryonic primary cortical neurons to compare the results with rat DIV14 embryonic primary cortical neurons.
Similarly,
To further confirm TATκ28-CDKL5 over time, the cultured embryonic primary cortical neurons were treated with 10 μg/ml recombinant TATκ28-CDKL5 for 15 min, 30 min, 2 hr, 6 hr, or 24 hours. At each timepoint, treated coverslips were fixed in 4% PFA, permeabilized in 0.1% saponin, and stained using anti-MAP2, anti-CDKL5, and/or anti-phosphorylated (S222) EB2 antibodies. The cells were counterstained with DAPI and mounted on glass microscope slides under Prolong Diamond anti-fade mounting medium. The samples were imaged using a Leica SP8 point scanning laser confocal microscope with a 63x oil-immersion objective. The images were processed using Leica Lightning software and merged and colorized using ImageJ software.
CDKL5 protein is reported to co-localize with PSD95 in neurons. In a particular embodiment, the DIV14 neurons were treated with 15 μg/ml of TATκ28-CDKL5 for 2 hours. The neurons were then stained with anti-PSD95 and anti-CDKL5.
SEQ ID NOS: 106-121 provide exemplary sequences for CDKL5 AAV vectors.
SEQ ID NO: 106 provides an exemplary sequence for a plasmid for expressing the full-length human CDKL5107 isoform using the CBh promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28. The DNA sequence is codon-optimized for expression in mice.
SEQ ID NO: 107 provides an exemplary sequence for a plasmid for expressing a kinase-dead version of the full-length human CDKL5107 isoform using the CBh promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28. The DNA sequence is codon-optimized for expression in mice.
SEQ ID NO: 108 provides an exemplary sequence for a plasmid for expressing eGFP using the CBh promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28. The DNA sequence is codon-optimized for expression in mice.
SEQ ID NO: 109 provides an exemplary sequence for a plasmid for expressing a fusion protein comprising NLS and eGFP using the CBh promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28. The DNA sequence is codon-optimized for expression in mice.
SEQ ID NO: 110 provides an exemplary sequence for a plasmid for expressing a fusion protein comprising a modified BiP leader signal polypeptide, TATκ28 and the full-length human CDKL5107 isoform using the CBh promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28. The DNA sequence is codon-optimized for expression in mice.
SEQ ID NO: 111 provides an exemplary sequence for a plasmid for expressing a fusion protein comprising a modified BiP leader signal polypeptide, TATκ28 and a kinase-dead version of the full-length human CDKL5107 isoform using the CBh promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28. The DNA sequence is codon-optimized for expression in mice.
SEQ ID NO: 112 provides an exemplary sequence for a plasmid for expressing a fusion protein comprising a modified BiP leader signal polypeptide, TATκ28 and eGFP using the CBh promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28. The DNA sequence is codon-optimized for expression in mice.
SEQ ID NO: 113 provides an exemplary sequence for a plasmid for expressing a fusion protein comprising a modified BiP leader signal polypeptide, TATκ28, NLS and eGFP using the CBh promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28. The DNA sequence is codon-optimized for expression in mice.
SEQ ID NO: 114 provides an exemplary sequence for a plasmid for expressing the full-length human CDKL5107 isoform using the hSyn1 promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28. The DNA sequence is codon-optimized for expression in mice.
SEQ ID NO: 115 provides an exemplary sequence for a plasmid for expressing a kinase-dead version of the full-length human CDKL5107 isoform using the hSyn1 promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28. The DNA sequence is codon-optimized for expression in mice.
SEQ ID NO: 116 provides an exemplary sequence for a plasmid for expressing eGFP using the hSyn1 promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28. The DNA sequence is codon-optimized for expression in mice.
SEQ ID NO: 117 provides an exemplary sequence for a plasmid for expressing a fusion protein comprising NLS and eGFP using the hSyn1 promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28. The DNA sequence is codon-optimized for expression in mice.
SEQ ID NO: 118 provides an exemplary sequence for a plasmid for expressing a fusion protein comprising a modified BiP leader signal polypeptide, TATκ28 and the full-length human CDKL5107 isoform using the hSyn1 promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28. The DNA sequence is codon-optimized for expression in mice.
SEQ ID NO: 119 provides an exemplary sequence for a plasmid for expressing a fusion protein comprising a modified BiP leader signal polypeptide, TATκ28 and a kinase-dead version of the full-length human CDKL5107 isoform using the hSyn1 promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28. The DNA sequence is codon-optimized for expression in mice.
SEQ ID NO: 120 provides an exemplary sequence for a plasmid for expressing a fusion protein comprising a modified BiP leader signal polypeptide, TATκ28 and eGFP using the hSyn1 promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28. The DNA sequence is codon-optimized for expression in mice.
SEQ ID NO: 121 provides an exemplary sequence for a plasmid for expressing a fusion protein comprising a modified BiP leader signal polypeptide, TATκ28, NLS and eGFP using the hSyn1 promoter and the L-ITR and R-ITR of SEQ ID NOS: 27 and 28. The DNA sequence is codon-optimized for expression in mice.
Plasmids containing SEQ ID NOS: SEQ ID NOS: 106-121 will be generated and tested in mice. Similar plasmids that are codon-optimized for rats will be tested in mice.
An exemplary DNA sequence codon-optimized for expression of a fusion protein in a human is provided in SEQ ID NO: 122. The fusion protein encoded by SEQ ID NO: 122 comprises a modified BiP leader signal polypeptide, TATκ28 and the full-length human CDKL5107 isoform.
An exemplary DNA sequence codon-optimized for expression of the full-length human CDKL5107 isoform in a human (but without the initiator methionine codon or the stop codon) is provided in SEQ ID NO: 123.
One skilled in the art can derive exemplary DNA sequences for human expression of the CDKL5 truncation variants described herein by deleting the relevant portions of the DNA sequence for the full-length CDKL5107 isoform.
Exemplary DNA sequences for the glycosylation variant fusion proteins of SEQ ID NOS: 93-105 that are codon-optimized for human expression are provided in SEQ ID NOS: 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146 and 148, respectively.
Exemplary DNA sequences for the glycosylation variant CDKL5 polypeptides of SEQ ID NOS: 13-25 that are codon-optimized for human expression (but without the initiator methionine codon or the stop codon) are provided in SEQ ID NOS: 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147 and 149, respectively.
Exemplary DNA sequences for TATκ11, TATκ28, Antennapedia, Transportan and P97 that are codon-optimized for human expression (but without the initiator methionine codon or the stop codon) are provided in SEQ ID NOS: 150-154, respectively. Exemplary DNA sequences for TATκκ28 that are codon-optimized for human expression (but without the initiator methionine codon or the stop codon) using different codon optimization tools are provided in SEQ ID NOS: 170-173
An exemplary DNA sequence for mBIP that is codon-optimized for human expression (including the initiator methionine codon but without the stop codon) is provided in SEQ ID NO: 155. An exemplary DNA sequence for mvBIP that is codon-optimized for human expression (including the initiator methionine codon but without the stop codon) is provided in SEQ ID NO: 169.
In this Example, CDKL5 null mice were used for determining BIP-TATκ28-CDKL5 induced cross-correction. The CDKL5 null mice were divided into a treatment group and a control group. The treatment group was administered AAV-PHP.B.CBH.BIP-TATκ28-CDKL5.SV40 through intracerebroventricular (ICV) injection in an amount of 10×e9 GC/mice or 10×e10 GC/mice. The control group mice were administered PBS. Three months post-administration, the impact of the vector on behavioral endpoints was assessed and the mice were euthanized for transgene expression analysis.
After euthanizing mice, sections of brain were taken. The sections were stained with DAPI, anti-NeuN antibody, anti-CDKL5 RNA riboprobe and anti-CDKL5 protein antibody.
An image analysis was performed using Visiopharm software and the cells were divided into six groups: (1) DAPI stain to identify cells; (2) NeuN stain to identify neurons; (3) Neurons having CDKL5 mRNA and CDKL5 protein; (4) Neurons having CDKL5 mRNA; and (5) Cross-corrected neurons.
An exemplary plasmid for expressing various fusion proteins is shown in
Reference throughout this specification to “one embodiment,” “certain embodiments,” “various embodiments,” “one or more embodiments” or “an embodiment” means that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of the phrases such as “in one or more embodiments,” “in certain embodiments,” “in various embodiments,” “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments.
Although the disclosure herein provided a description with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the present disclosure without departing from the spirit and scope thereof. Thus, it is intended that the present disclosure include modifications and variations that are within the scope of the appended claims and their equivalents.
ESFLNGNLAGASLSP
VSTRVSSLPSESSSGT
HSKRQPAF
ESFLNGNLAGASLSP
VSTRVSSLPSESSSGT
HSKRQPAF
ESFLNGNLAGASLSP
VSTRVSSLPSESSSGT
HSKRQPAF
ESFLNGNLAGASLSP
VSTRVSSLPSESSSGT
HSKRQPAF
MKLSLVAAMLLLLSLVAAMLLLLSAARAGDAAQPARRARRTKLAAYARKAARQARAGGGGSKI
METDTLLLWVLLLWVPGSTGGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMN
MKLSLVAAMLLLLSLVAAMLLLLSAARAGDAAQPARRARRTKLAAYARKAARQARAGGGGSKI
METDTLLLWVLLLWVPGSTGGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMN
MGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKC
DHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
DHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
HH*
HHHH*
DHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
H*
YKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
MKLSLVAAMLLLLSLVAAMLLLLSAARAGYARKAARQARAGGGGSKIPNIGNVMNKFEILGW
METDTLLLWVLLLWVPGSTGGYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVV
DYKDHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
MGYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKD
DDKDGAPHHHHHH*
DDKDGAPHHHHHH*
DDKDGAPHHHHHH*
MGYGRKKRRQRRRGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKD
DDKDGAPHHHHHH
MGRQIKIWFQNRRMKWKKGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAI
DYKDDDDKDGAPHHHHHH*
MGAGYLLGKINLKALAALAKKILGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETH
KDHDIDYKDDDDKDGAPHHHHHH*
MGDAAQPARRARRTKLAAYGRKKRRQRRRGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKC
DHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
MKLSLVAAMLLLLSLVAAMLLLLSAARAGDSSHAFTLDELRGGGGSKIPNIGNVMNKFEILGV
MGDSSHAFTLDELRGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFK
DDDKDGAPHHHHHH*
MGDAAQPARRARRTKLAAYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKC
DHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
MGYARKAARQARAGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKD
DDKDGAPHHHHHH*
MGDAAQPARRARRTKLAAYGRKKRRQRRRGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKC
DHDGDYKDHDIDYKDDDDKDGAPHHHHHH*
MGYGRKKRRQRRRGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKD
DDKDGAPHHHHHH*
MGRQIKIWFQNRRMKWKKGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAI
DYKDDDDKDGAPHHHHHH*
MGAGYLLGKINLKALAALAKKILGGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETH
KDHDIDYKDDDDKDGAPHHHHHH*
MKLSLVAAMLLLLSLVAAMLLLLSAARAGDAAQPARRARRTKLAAYARKAARQARAGGGGSKI
M
SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDV
KLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKL
PEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQI
DKYLKSSKYIAWPLQGWQATFGGGDHPPKSGGGGSLEVLFQGPDAAQPARRARRTKLAAYARK
AARQARA
GGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEE
M
SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDV
KLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKL
PEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQI
DKYLKSSKYIAWPLQGWQATFGGGDHPPKSGGGGSLEVLFQGPYARKAARQARAGGGGSKIPN
M
SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDV
KLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKL
PEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQI
DKYLKSSKYIAWPLQGWQATFGGGDHPPKSGGGGSLEVLFQGPDAAQPARRARRTKLAAYGRK
KRRQRRR
GGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEE
M
SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDV
KLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKL
PEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQI
DKYLKSSKYIAWPLQGWQATFGGGDHPPKSGGGGSLEVLFQGPYGRKKRRQRRRGGGGSKIPN
M
SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDV
KLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKL
PEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQI
DKYLKSSKYIAWPLQGWQATFGGGDHPPKSGGGGSLEVLFQGPRQIKIWFQNRRMKWKKGGGG
M
SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDV
KLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKL
PEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQI
DKYLKSSKYIAWPLQGWQATFGGGDHPPKSGGGGSLEVLFQGPAGYLLGKINLKALAALAKKI
L
GGGGSKIPNIGNVMNKFEILGVVGEGAYGVVLKCRHKETHEIVAIKKFKDSEENEEVKETTL
HH*
M
SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDV
KLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKL
PEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQI
DKYLKSSKYIAWPLQGWQATFGGGDHPPKSGGGGSLEVLFQGPDSSHAFTLDELRGGGGSKIP
M
SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDV
KLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKL
PEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQI
DKDGAPHHHHHH*
M
SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDV
KLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKL
PEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQI
M
SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDV
KLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKL
PEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQI
DKYLKSSKYIAWPLQGWQATFGGGDHPPKSGGGGSLEVLFQGPGKIPNIGNVMNKFEILGVVG
MKLSLVAAMLLLLSLVAAMLLLLSAARAGDAAQPARRARRTKLAAYARKAARQARAGGGGSKI
MKLSLVAAMLLLLSLVAAMLLLLSAARAGDAAQPARRARRTKLAAYARKAARQARAGGGGSKI
MKLSLVAAMLLLLSLVAAMLLLLSAARAGDAAQPARRARRTKLAAYARKAARQARAGGGGSKI
MKLSLVAAMLLLLSLVAAMLLLLSAARAGDAAQPARRARRTKLAAYARKAARQARAGGGGSKI
MKLSLVAAMLLLLSLVAAMLLLLSAARAGDAAQPARRARRTKLAAYARKAARQARAGGGGSKI
MKLSLVAAMLLLLSLVAAMLLLLSAARAGDAAQPARRARRTKLAAYARKAARQARAGGGGSKI
MKLSLVAAMLLLLSLVAAMLLLLSAARAGDAAQPARRARRTKLAAYARKAARQARAGGGGSKI
MKLSLVAAMLLLLSLVAAMLLLLSAARAGDAAQPARRARRTKLAAYARKAARQARAGGGGSKI
MKLSLVAAMLLLLSLVAAMLLLLSAARAGDAAQPARRARRTKLAAYARKAARQARAGGGGSKI
ESFLNGNLAGASLSPLHT
VSTRVSSLPSESSSGT
HSKRQPAFDPW
ESFLNGNLAGASLSPLHT
VSTRVSSLPSESSSGT
HSKRQPAFDPW
ESFLNGNLAGASLSPLHT
VSTRVSSLPSESSSGT
HSKRQPAFDPW
ESFLNGNLAGASLSPLHT
VSTRVSSLPSESSSGT
HSKRQPAFDPW
ATGAAGCTGTCCCTGGTGGCCGCTATGCTGCTGCTGCTGTCTCTGGTCGCTGCCATGTTATTA
CTGCTGTCTGCCGCTAGGGCCGGGGACGCAGCACAGCCCGCAAGAAGAGCAAGAAGAACTAAA
CTGGCCGCTTACGCAAGGAAGGCAGCAAGACAGGCAAGAGCAGGCGGCGGCGGCTCCAAGATC
CTGGCCGCTTACGCAAGGAAGGCAGCAAGACAGGCAAGAGCAGGCGGCGGCGGCTCCAAGATC
CTGGCCGCTTACGCAAGGAAGGCAGCAAGACAGGCAAGAGCAGGCGGCGGCGGCTCCAAGATC
CTGGCCGCTTACGCAAGGAAGGCAGCAAGACAGGCAAGAGCAGGCGGCGGCGGCTCCAAGATC
CTGGCCGCTTACGCAAGGAAGGCAGCAAGACAGGCAAGAGCAGGCGGCGGCGGCTCCAAGATC
CTGGCCGCTTACGCAAGGAAGGCAGCAAGACAGGCAAGAGCAGGCGGCGGCGGCTCCAAGATC
CTGGCCGCTTACGCAAGGAAGGCAGCAAGACAGGCAAGAGCAGGCGGCGGCGGCTCCAAGATC
CTGGCCGCTTACGCAAGGAAGGCAGCAAGACAGGCAAGAGCAGGCGGCGGCGGCTCCAAGATC
CTGGCCGCTTACGCAAGGAAGGCAGCAAGACAGGCAAGAGCAGGCGGCGGCGGCTCCAAGATC
CTGGCCGCTTACGCAAGGAAGGCAGCAAGACAGGCAAGAGCAGGCGGCGGCGGCTCCAAGATC
CTGGCCGCTTACGCAAGGAAGGCAGCAAGACAGGCAAGAGCAGGCGGCGGCGGCTCCAAGATC
GAGAGCTTCCTGAATGGAAACCTGGCAGGAGCCTCTCTGAGCCCACTGCACACA
GTGAGCACCCGGGTGAGCAGC
CACTCTAAGAGGCAGCCCGCCTTTGACCCTTGG
GAGAGCTTCCTGAATGGAAACCTGGCAGGAGCCTCTCTGAGCCCACTG
GTGAGCACCCGGGTG
CACTCTAAGAGGCAGCCCGCCTTTGAC
CTGGCCGCTTACGCAAGGAAGGCAGCAAGACAGGCAAGAGCAGGCGGCGGCGGCTCCAAGATC
GAGAGCTTCCTGAATGGAAACCTGGCAGGAGCCTCTCTGAGCCCACTGCACACA
GTGAGCACCCGGGTGAGCAGC
CACTCTAAGAGGCAGCCCGCCTTTGACCCTTGG
GAGAGCTTCCTGAATGGAAACCTGGCAGGAGCCTCTCTGAGCCCACTG
GTGAGCACCCGGGTG
CACTCTAAGAGGCAGCCCGCCTTTGAC
CTGGCCGCTTACGCAAGGAAGGCAGCAAGACAGGCAAGAGCAGGCGGCGGCGGCTCCAAGATC
GAGAGCTTCCTGAATGGAAACCTGGCAGGAGCCTCTCTGAGCCCACTGCACACA
GTGAGCACCCGGGTGAGCAGC
CACTCTAAGAGGCAGCCCGCCTTTGACCCTTGG
GAGAGCTTCCTGAATGGAAACCTGGCAGGAGCCTCTCTGAGCCCACTG
GTGAGCACCCGGGTG
CACTCTAAGAGGCAGCCCGCCTTTGAC
CTGGCCGCTTACGCAAGGAAGGCAGCAAGACAGGCAAGAGCAGGCGGCGGCGGCTCCAAGATC
GAGAGCTTCCTGAATGGAAACCTGGCAGGAGCCTCTCTGAGCCCACTGCACACA
GTGAGCACCCGGGTGAGCAGC
CACTCTAAGAGGCAGCCCGCCTTTGACCCTTGG
GAGAGCTTCCTGAATGGAAACCTGGCAGGAGCCTCTCTGAGCCCACTG
GTGAGCACCCGGGTG
CACTCTAAGAGGCAGCCCGCCTTTGAC
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/58247 | 10/30/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62928134 | Oct 2019 | US | |
| 62928140 | Oct 2019 | US | |
| 63090520 | Oct 2020 | US |
