Patent Application
20220281930
This application contains a Sequence Listing submitted as an electronic text file named “05_20-1246-WO_Sequence-Listing_ST25.txt”, having a size in bytes of 114 kb, and created on Aug. 24, 2020. The information contained in this electronic file is hereby incorporated by reference in its entirety.
The present disclosure provides compositions and methods for the treatment of pathological pain and itch.
Inhibitory GABA-ergic neurotransmission is of fundamental relevance for the adult vertebrate central nervous system and requires low chloride ion concentration in neurons. In the mature vertebrate central nervous system (CNS), γ-aminobutyric acid (GABA) acts primarily as an inhibitory neurotransmitter that is critical for normal CNS functioning. In chronic pain and itch, GABA-ergic transmission is compromised, causing circuit malfunction and disrupting inhibitory networks. Therefore, the need arises to discover new approaches to restore physiologic GABA-ergic transmission. This would increase the basic understanding of these sensory disorders to address the unmet medical need of chronic pain and itch, with safer and more effective alternatives to opioids for chronic pain.
In the adult vertebrate CNS, the K+/Cl− cotransporter (KCC2) is expressed in neurons, continuously extruding chloride ions, thus ensuring that intracellular levels of chloride ions remain low, which is essential for inhibitory GABA-ergic neurotransmission. In chronic pathologic pain and other neuro-psychiatric illnesses, KCC2 expression is attenuated in the primary sensory gate in spinal cord dorsal horn neurons. This process is one key pathophysiological mechanism that contributes to an excitation/inhibition imbalance, more specifically it corrupts inhibitory neurotransmission and causes inhibitory circuit malfunction. Notably, there is no “back-up” protein that can rescue the KCC2 expression deficit.
Thus, there is a need for novel therapies and methods to treat chronic pain by rescuing the KCC2 expression deficit.
The present disclosure is based, in part, on the discovery by the inventor that renormalizing inhibitory neurotransmission can be achieved by upregulating neuronal chloride-extruding transporter, KCC2 (SLC12A5), via GSK3ß inhibition and/or by use of a gene-therapeutic approach leveraging a novel signaling mechanism of delta-2 catenin.
One aspect of the present disclosure provides a recombinant transgene that includes a polynucleotide that encodes human-delta-catenin protein (e.g., SEQ ID NO:20), or a human-delta-catenin protein variant, including human-delta-catenin (S276A) protein (SEQ ID NO:32) or a fragment, isoform, or homologue thereof.
An aspect of the disclosure provides an expression cassette including a nucleotide sequence encoding the human-delta-catenin protein (e.g., SEQ ID NO:20), or a human-delta-catenin protein variant, including human-delta-catenin (S276A) protein (SEQ ID NO:32) or a fragment, isoform, or homologue thereof.
In some embodiments, the expression cassette further includes a nucleotide sequence that is codon-optimized to reduce CpG methylation sites and mammalian expression encoding the human-delta-catenin transgene. In some embodiments, the expression cassette includes a human-delta-catenin transgene sequence operably linked to a promoter and a polyadenylation sequence, which, in some embodiments, includes synapsin 1, calcium/calmodulin-dependent protein kinase II, tubulin alpha 1, neuron-specific enolase, human KCC2 promoter, or platelet-derived growth factor beta chain promoters.
In some embodiments, the expression cassette includes a constitutively active promoter, which, in some embodiments, includes human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, and herpes simplex virus thymidine kinase. In some embodiments, the expression cassette includes a neuro-specific promoter.
In some embodiments, the expression cassette further includes a transcriptional termination signal, which signal can include bovine growth hormone polyadenylation signal (BGHpA), Simian virus 40 polyadenylation signal (SV40pA), and a synthetic polyadenylation signal.
An aspect of the disclosure provides a recombinant viral vector including a nucleotide sequence encoding the gene for human-delta-catenin (e.g., SEQ ID NO:20), or a human-delta-catenin protein variant, including human-delta-catenin (S276A) protein (SEQ ID NO:32), or a fragment, isoform, or homologue thereof. In some embodiments, the recombinant viral vector includes an expression cassette as described above and herein. In some embodiments, the recombinant viral vector further includes one or more of the following elements: (a) an inverted terminal repeat sequence (ITR); (b) a promoter; (c) an intron; (d) transcription terminator; and (e) a flanking inverted terminal repeat sequence (ITR).
In some embodiments of the recombinant viral vector that include a promoter, the promoter can include synapsin 1, calcium/calmodulin-dependent protein kinase II, tubulin alpha 1, neuron-specific enolase, human KCC2 promoter, or platelet-derived growth factor beta chain promoters. In some embodiments, the promoter is a constitutively active promoter, which, in some embodiments, can include human β-actin, human elongation factor-1a, chicken 3-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, and herpes simplex virus thymidine kinase promoters. In some embodiments, the recombinant viral vector includes a neuro-specific promoter.
In some embodiments, the recombinant viral vector further includes a transcriptional termination signal, which can include bovine growth hormone polyadenylation signal (BGHpA), Simian virus 40 polyadenylation signal (SV40pA), and a synthetic polyadenylation signal.
In some embodiments, the recombinant viral vector can include adenoviruses, Adeno-associated viruses (AAV), Herpes simplex viruses (e.g., Herpes Simplex Virus Type 1), Retroviruses, lentiviruses, alphaviruses, flaviviruses, rhabdoviruses, measles virus, Newcastle disease virus, poxviruses, and picornaviruses. In some embodiments, the recombinant viral vector is an adeno-associated virus (AAV).
In some embodiments, the recombinant AAV vector can include a serotype of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.
In another aspect the disclosure provides compositions including a transgene as described above and herein, an expression cassette as described above and herein, or a recombinant viral vector as described above and herein. In some embodiments, the compositions are pharmaceutical compositions that include a pharmaceutically acceptable carrier and/or excipient.
In another aspect, the disclosure provides methods related to the transgenes, expression cassettes, recombinant viral vectors, recombinant AAV vectors, compositions and pharmaceutical compositions described above and herein. In some embodiments, methods of treating pain in a subject in need thereof are provided, including administering a therapeutically effective amount of a composition as described above and herein such that the pain is treated in the subject. In some embodiments, the pain is neuropathic pain or pathological pain. In some embodiments, methods of treating itch in a subject are provided, including administering to the subject a therapeutically effective amount of a composition described above and herein such that the itch is treated in the subject. In some embodiments, the itch is neuropathic or pathological itch.
In some embodiments, methods of increasing KCC2 mRNA levels in a subject are provided, including administering to the subject a therapeutically effective amount of a composition as described above and herein so that the KCC2 mRNA levels are increased in the subject compared to a pre-treatment baseline.
In some embodiments, methods of reducing intracellular chloride ion levels ([Cl—]i) in a central nervous system cell in a subject are provided, including administering to the subject a therapeutically effective amount of a composition as described above and herein so that the [Cl—]i in a central nervous system cell is reduced in the subject compared to a pre-treatment baseline.
In some embodiments, methods of increasing chloride ion efflux in a central nervous system cell in a subject are provided, including administering to the subject a therapeutically effective amount of a composition as described above and herein so that the chloride ion efflux is increased in the subject.
In some embodiments, methods of increasing synaptophysin expression in a central nervous system cell in a subject are provided, including administering to the subject a therapeutically effective amount of a composition as described above and herein so that the synaptophysin expression is increased in the subject as compared to a pre-treatment baseline.
In some embodiments, methods of increasing KCC2 expression in a subject are provided, including administering to the subject a therapeutically effective amount of a composition as described above and herein so that KCC2 expression is increased in the subject compared to pre-treatment baseline.
In some embodiments of the methods described above and herein, the methods further include administering to the subject a therapeutically effective amount of at least one additional compound, which compound, in some embodiments, includes a GSK3β inhibitor, an anti-analgesic, a muscle relaxant, an anti-anxiety drug, an antidepressant, an anticonvulsant, and combinations thereof. In some embodiments, the at least one additional compound includes a GSK3β inhibitor a corticosteroid, a counterirritant, an antihistamine, and a local anesthetic and combinations thereof.
In another aspect, the disclosure provides methods of treating pain and/or treating itch in a subject in need thereof, including administering a therapeutically effective amount of a GSK3β inhibitor, or a pharmaceutical composition including a GSK3β inhibitor and a pharmaceutically acceptable carrier and/or excipient, such that the pain is treated in the subject. In some embodiments, the pain and/or itch is neuropathic or pathological in nature.
In some embodiments, methods of increasing KCC2 mRNA levels in a subject are provided, including administering to the subject a therapeutically effective amount of a GSK3β inhibitor, or a pharmaceutical composition including a GSK3β inhibitor and a pharmaceutically acceptable carrier and/or excipient, so that the KCC2 mRNA levels are increased in the subject.
In some embodiments, methods of reducing intracellular chloride ion levels ([Cl—]i) in a central nervous system cell in a subject are provided, including administering to the subject a therapeutically effective amount of a GSK3β inhibitor, or a pharmaceutical composition including a GSK3β inhibitor and a pharmaceutically acceptable carrier and/or excipient, so that the [Cl—]i in a central nervous system cell is reduced in the subject.
In some embodiments, methods of increasing chloride ion efflux in a central nervous system cell in a subject are provided, including administering to the subject a therapeutically effective amount of a GSK3β inhibitor, or a pharmaceutical composition including a GSK3β inhibitor and a pharmaceutically acceptable carrier and/or excipient, so that the chloride ion efflux is increased in the subject.
In some embodiments, methods of increasing synaptophysin expression in a central nervous system cell in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a GSK3β inhibitor, or a pharmaceutical composition comprising a GSK3β inhibitor and a pharmaceutically acceptable carrier and/or excipient, such that the synaptophysin expression is increased in the subject.
In some embodiments, methods of increasing KCC2 expression in a subject are provided, including administering to the subject a therapeutically effective amount of a GSK3β inhibitor, or a pharmaceutical composition including a GSK3β inhibitor and a pharmaceutically acceptable carrier and/or excipient, so that KCC2 expression is increased in the subject.
In some embodiments of the methods described above, the methods further include administering to the subject a therapeutically effective amount of at least one additional compound, which compound, in some embodiments, includes compositions including a transgene, an expression cassette, or a recombinant viral vector as described above and herein (each, in some embodiments, pharmaceutical compositions that include a pharmaceutically acceptable carrier and/or excipient), an anti-analgesic, a muscle relaxant, an anti-anxiety drug, an antidepressant, an anticonvulsant, and combinations thereof. In some embodiments, the additional compound can include compositions including a transgene, an expression cassette, or a recombinant viral vector as described above and herein (each, in some embodiments, pharmaceutical compositions that include a pharmaceutically acceptable carrier and/or excipient), a corticosteroid, a counterirritant, an antihistamine, and a local anesthetic and combinations thereof.
In the methods described in all relevant aspects and embodiments of the disclosure, the central nervous system cell is a neuron.
In the methods described in all relevant aspects and embodiments of the disclosure, the composition can be administered by any administration route, including intrathecally, intra-cerebroventricularly, intra-cerebrally, perispinally, intra-spinally, intravenously and others.
LUC. N2a differentiated cells were used. Top panel is a schematic of the mouse KCC2 promoter constructs, as in
For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.
Articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.
“About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result.
The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).
As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”
Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise-indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.
As used herein, the term “nucleic acid” refers to isolated, purified, natural, recombinant, synthetic deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the reference sequence explicitly indicated.
As used herein, “nucleic acid therapy” or “gene therapy” refers to the transfer or insertion of nucleic acid molecules into certain cells, which may be referred to as target cells, to produce specific gene products that are involved in correcting or modulating diseases or disorders and/or promote beneficial biological processes. The nucleic acid is introduced into the selected target cells in a manner such that the nucleic acid is expressed and a product encoded thereby is produced. Alternatively, the nucleic acid may in some manner mediate the expression of nucleic acid that encodes a therapeutic product. This product may be a therapeutic compound, which is produced in therapeutically effective amounts or at a therapeutically useful time. It may also encode a product, such as a peptide or RNA, that in some manner mediates, directly or indirectly, expression of a therapeutic product. Expression of the nucleic acid by the target cells within an organism afflicted with a disease or disorder thereby provides a way to modulate the disease or disorder or beneficial biological processes. The nucleic acid encoding the therapeutic product may be modified prior to introduction into the target cell in order to enhance or otherwise alter the product or expression thereof. Nucleic acid therapy also refers to administration or in situ generation of a nucleic acid or a derivative thereof which specifically hybridizes (e.g., binds) under cellular conditions with the cellular mRNA and/or genomic DNA encoding one of a target polypeptides so as to inhibit production of that protein, e.g., by inhibiting transcription and/or translation. The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.
For use in nucleic acid therapy, cells can be transfected in vitro via methods of the disclosure, followed by introduction of the transfected cells into the body of a subject. This is often referred to as ex vivo nucleic acid therapy. Alternatively, the cells can be transfected directly in vivo within the body of a subject.
As used herein, “heterologous” or “foreign” with reference to nucleic acids, DNA and RNA are used interchangeably and refer to nucleic acid, DNA or RNA that does not occur naturally as part of the genome in which it is present or which is found in a location(s) or in an amount in the genome that differs from that in which it occurs in nature. It is nucleic acid that has been exogenously introduced into the cell. Thus, heterologous nucleic acid is nucleic acid not normally found in the host genome in an identical context. Examples of heterologous nucleic acids include, but are not limited to, DNA that encodes a gene product or gene product(s) of interest, introduced for purposes of gene therapy or for production of an encoded protein. Other examples of heterologous DNA include, but are not limited to, DNA that encodes a selectable marker, DNA that encodes therapeutically effective substances, such as anti-pain or anti-itch agents, enzymes and hormones, and DNA that encodes other types of proteins, such as antibodies.
As used herein, the term “promoter” refers to a DNA regulatory region capable of binding RNA polymerase in a mammalian cell and initiating transcription of a downstream (3′ direction) coding sequence operably linked thereto. For purposes of the present disclosure, a promoter sequence includes the minimum number of bases or elements necessary to initiate transcription of a gene of interest at levels detectable above background. Within the promoter sequence may be a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. Promoters include those that are naturally contiguous to a nucleic acid molecule and those that are not naturally contiguous to a nucleic acid molecule. Additionally, the term “promoter” includes inducible promoters, conditionally active promoters such as a cre-lox promoter, constitutive promoters, and tissue specific promoters. Examples of suitable promoter include, but are not limited to, synapsin 1, calcium/calmodulin-dependent protein kinase II, tubulin alpha 1, neuron-specific enolase, human KCC2 promoter, platelet-derived growth factor beta chain promoters and the like.
As used herein, “transformation” or “transfection” or “transfected” refers to the process by which nucleic acids are introduced into cells with or without the use of one or more accompanying facilitating agents such as lipofectamine. Transfection refers to the taking up of exogenous nucleic acid, by a host cell whether or not any coding sequences are in fact expressed. Methods and compositions of the disclosure are effective for transformation or transfection. Successful transfection is generally recognized by detection of the presence of the heterologous nucleic acid within the transfected cell, such as, for example, any visualization of the heterologous nucleic acid or any indication of the operation of a such nucleic acid within the host cell. Methods for transfection that are known in the art include, e.g., calcium phosphate transfection, DEAE dextran transfection, protoplast fusion, electroporation, and lipofection.
As used herein, the term “expression” refers to the conversion of the information contained in the nucleic acid molecule into a gene product. The gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a peptide or polypeptide produced by translation of an mRNA. Gene products also include RNAs that are modified by processes such as capping, polyadenylation, methylation, and editing; and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.
As used herein, the term “host cell” refers to an individual cell or a cell culture that can be or has been a recipient of any recombinant vector(s) or isolated polynucleotide(s). Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention. A host cell that comprises a recombinant vector of the invention may be called a “recombinant host cell.”
As used herein, the term “recombinant,” with respect to a nucleic acid molecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic, and/or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term “recombinant”, as used with respect to a protein or polypeptide, means a polypeptide produced by expression of a recombinant polynucleotide. The term “recombinant” as used with respect to a host cell means a host cell into which a recombinant polynucleotide has been introduced.
As used herein, the phrase “recombinant virus” and “recombinant viral vector” are used interchangeable and refer to a virus or viral vector that is genetically modified by the hand of man. The phrase covers any virus known in the art. In some embodiments, the recombinant viral vector is selected from the group consisting of adenoviruses, Adeno-associated viruses (AAV), Herpes simplex viruses (e.g., Herpes Simplex Virus Type 1), Retroviruses, lentiviruses, alphaviruses, flaviviruses, rhabdoviruses, measles virus, Newcastle disease virus, poxviruses, and picornaviruses. As used herein, the term “vector” refers to an agent (e.g., a plasmid or virus) used to transmit genetic material to a host cell or organism. A vector may be composed of either DNA or RNA. In some embodiments, the recombinant viral vector comprises a recombinant AAV vector. In other embodiments, recombinant AAV vector comprises a serotype selected from the group consisting of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV 11, AAV12, or AAV13. In some embodiments, the recombinant AAV vector is selected from the group consisting of AAV1, AAV8, and AAV9. In certain embodiments, the recombinant AAV vector comprises AAV9. In other embodiments, the virus comprises a lentivirus.
As used herein, the term “biologically active” entity, or an entity having “biological activity,” is one having structural, regulatory, or biochemical functions of a naturally occurring molecule or any function related to or associated with a metabolic or physiological process. Biologically active polynucleotide fragments are those exhibiting activity similar, but not necessarily identical, to an activity of a polynucleotide of the present invention. The biological activity can include an improved desired activity, or a decreased undesirable activity. For example, an entity demonstrates biological activity when it participates in a molecular interaction with another molecule, such as hybridization, when it has therapeutic value in alleviating a disease condition, when it has prophylactic value in inducing an immune response, when it has diagnostic and/or prognostic value in determining the presence of a molecule, such as a biologically active fragment of a polynucleotide that can, for example, be detected as unique for the polynucleotide molecule, or that can be used as a primer in a polymerase chain reaction. A biologically active polypeptide or fragment thereof includes one that can participate in a biological reaction.
As used herein, the terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, and the like. Furthermore, a “polypeptide” may refer to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate or may be accidental.
As described herein, “sequence identity” is related to sequence homology. Homology comparisons may be conducted by eye or using sequence comparison programs. These commercially available computer programs may calculate percent (%) homology between two or more sequences and may also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences. Sequence homologies may be generated by any of a number of computer programs known in the art, for example BLAST or FASTA.
Percentage (%) sequence identify can be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Ungapped alignments are performed only over a relatively short number of residues. Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion may cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in percent homology when a global alignment is performed. Therefore, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without unduly penalizing the overall homology or identity score. This is achieved by inserting “gaps” in the sequence alignment to try to maximize local homology or identity.
As used herein, “administration” of a disclosed compound encompasses the delivery to a subject of a compound as described herein, or a prodrug or other pharmaceutically acceptable derivative thereof, using any suitable formulation or route of administration, as discussed herein.
As used herein, the terms “effective amount” or “therapeutically effective amount” refer to that amount of a compound, transgene, and any pharmaceutical compositions thereof and described herein that is sufficient to effect the intended application including, but not limited to, disease treatment, as illustrated below. In some embodiments, the amount is that effective for detectable reduction of pain or itch. In some embodiments, the amount is that effective for alleviating, reducing or eliminating a pathologic pain or itch condition.
The therapeutically effective amount can vary depending upon the intended application, or the subject and disease condition being treated, e.g., the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the weight and age of the patient, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells, e.g., reduction of cell migration. The specific dose will vary depending on, for example, the particular compounds chosen, the species of subject and their age/existing health conditions or risk for health conditions, the dosing regimen to be followed, the severity of the disease, whether it is administered in combination with other agents, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.
For a recombinant viral vector, e.g., rAAV, an effective amount is also an amount sufficient to infect a sufficient number of cells of a target tissue in a subject. In some cases, an effective amount of a rAAV may be an amount sufficient to produce a stable somatic transgenic animal model. As an example regarding the effects of route of administration, targeting a CNS tissue by intravascular injection may require different (e.g., higher) doses, in some cases, than targeting CNS tissue by intrathecal or intracerebral injection. In some cases, multiple doses of a rAAV may be administered.
As used herein, “treatment,” “therapy” and/or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition.
As used herein, the term “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals. The term “nonhuman animals” of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. In some embodiments, the subject comprises a human. In other embodiments, the subject comprises a human suffering from pain and/or itch.
As used herein, the term “disease” and “disorder” are used interchangeably and refer to any disorder or structure or function is a human, animal, or plant, especially one that produces signs or symptoms or that affects a specific location and is not simply a direct result of physical injury. Examples include, but are not limited to, neurological disorders, pain, itch, metabolic disorders, cancer, genetic disorders, and the like.
As used herein, the term “pain” refers to the basic bodily sensation induced by a noxious stimulus, received by naked nerve endings, characterized by physical discomfort (e.g., pricking, throbbing, aching, etc.) and typically leading to an evasive action by the individual. As used herein, the term pain also includes chronic and acute neuropathic pain as well as pathologic pain. The terms “neuropathic pain” or “neurogenic pain” can be used interchangeable and refer to pain that arises from direct stimulation of nervous tissue itself, central or peripheral and can persist in the absence of stimulus. The sensations that characterize neuropathic pain vary and are often multiple and include burning, gnawing, aching, and shooting. (See Rooper and Brown, (2005) Adams and Victor's Principles of Neurology, 8th ed., NY, McGraw-Hill). These damaged nerve fibers send incorrect signals to other pain centers. The impact of nerve fiber injury includes a change in nerve function both at the site of injury and areas around the injury. Chronic neuropathic pain often seems to have no obvious cause; however, some common causes may include, but are not limited to, alcoholism, amputation, back, leg and hip problems, chemotherapy, diabetes, facial nerve problems, HIV infection or AIDS, multiple sclerosis, shingles, and spine surgery. For example, one example of neuropathic pain is phantom limb syndrome, which occurs when an arm or leg has been removed because of illness or injury, but the brain still gets pain messages from the nerves that originally carried impulses from the missing limb. The term “pathologic pain” refers to that pain that is characterized by an amplified response to normally innocuous stimuli, and an amplified response to acute pain. Pain as used herein may also refer to both chronic and acute pain. Suitable examples of pain include, but are not limited to, pain of osteoarthritis, cancer pain, chronic low back pain, low back pain of osteoporosis, pain of bone fracture, pain of rheumatoid arthritis, neuropathic pain, postherpetic pain, pain of diabetic neuropathy, fibromyalgia, pain of pancreatitis, pain of interstitial cystitis, pain of endometriosis, pain of irritable bowel syndrome, migraine, postoperative pain, pain of pulpitis and the like.
As used herein, the terms “itch,” “pruritus” and its alternative spelling “pruritis” are used interchangeably and refer to those irritating skin sensations that provoke a desire to scratch. Pruritus may range from mildly unpleasant and temporary to acute and persistent sensations. A number of skin (e.g., fungal infections, and skin conditions such as atopic dermatitis) and systemic conditions (e.g., renal failure, liver damage, liver disease (e.g., cirrhosis), acquired immune deficiency syndrome (AIDS), polycythemia vera, diabetes, hyperthyroidism, and cancer (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, and Kaposi's sarcoma) may be associated with acute and/or chronic pruritus, which can significantly reduce quality of life. Other causes of pruritus may include induction by cytokines or treatments such as chemotherapy and kidney dialysis. Suitable examples of pruritus may include, but are not limited to, systemic cutaneous pruritus, localized cutaneous pruritus, senile cutaneous pruritus, gestational pruritus, pruritus ani, vulvar pruritus and the like.
As used herein, the term “expression cassette” refers to a component of vector DNA consisting of a gene and regulatory sequence to be expressed by a transfected cell. In each successful transformation, the expression cassette directs the cell's machinery to make RNA and protein(s).
As used herein, the term “central nervous system cell” refers to neurons.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
Compositions
The present disclosure is based, in part, on the discovery by the inventor that renormalizing inhibitory neurotransmission can be achieved by upregulating neuronal chloride-extruding transporter, KCC2 (SLC12A5), via GSK3ß inhibition and/or by use of a gene-therapeutic approach leveraging a novel signaling mechanism of delta-2 catenin.
Accordingly, one aspect of the disclosure provides a recombinant transgene that includes a polynucleotide that encodes human-delta-catenin protein, or a variant thereof. In some embodiments, the polynucleotide can comprise SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, or a sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30. In some embodiments, the polynucleotide encodes human-delta-catenin (S276A), or a variant thereof including polynucleotides that code for conservative substitutions of alanine, such as glycine (S276G), valine (S276V), leucine (S276L) or isoleucine (S276I). In some embodiments, the polynucleotide comprises SEQ ID NO:32, and in some embodiments, the polynucleotide include any that codes for the polypeptide of SEQ ID NO:33, or a sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:32 and SEQ ID NO:33.
Another aspect of the disclosure provides a nucleic acid expression cassette that includes a nucleic acid sequence encoding the human delta-catenin protein, or a variant thereof. In some embodiments, the nucleic acid sequence in the expression cassette includes SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, or a sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30. In some embodiments, the nucleic acid sequence encodes human-delta-catenin (S276A), or a variant thereof including polynucleotides that code for conservative substitutions of alanine, such as glycine (S276G), valine (S276V), leucine (S276L) or isoleucine (S276I). In some embodiments, the polynucleotide includes SEQ ID NO:32 or a sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:32.
In some embodiments, the expression cassette further includes a nucleotide sequence that is codon-optimized to reduce CpG methylation sites and mammalian expression encoding the human-delta-catenin transgene.
In some embodiments, the cassette comprises the human delta-catenin protein operably linked to a promoter and a polyadenylation sequence. Any promoter suitable for the expression of human-delta-catenin protein may be used. Examples include, but are not limited to, synapsin 1, calcium/calmodulin-dependent protein kinase II, tubulin alpha 1, neuron-specific enolase, human KCC2 promoter, platelet-derived growth factor beta chain promoters, and the like.
In some embodiments, the promoter includes a constitutively active promoter. In some embodiments, the constitutively active promoter can be, for example, human β-actin, human elongation factor-1a, chicken 3-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, and herpes simplex virus thymidine kinase. In some embodiments, the expression cassette includes a neuro-specific promoter.
In some embodiments, the nucleic acid expression cassette further includes a transcriptional termination signal selected from the group consisting of bovine growth hormone polyadenylation signal (BGHpA), Simian virus 40 polyadenylation signal (SV40pA), and a synthetic polyadenylation signal.
Some embodiments of recombinant transgenes, expression cassettes, recombinant viral vectors and recombinant AAV vectors of the disclosure further include a transcriptional termination signal selected from the group consisting of bovine growth hormone polyadenylation signal (BGHpA), Simian virus 40 polyadenylation signal (SV40pA), and a synthetic polyadenylation signal.
Another aspect of the present disclosure provides compositions including a recombinant transgene that includes a polynucleotide that encodes human-delta-catenin protein, or a variant thereof as described herein. In some embodiments, the polynucleotide can comprise SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, or a sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30. In some embodiments, the nucleic acid sequence encodes human-delta-catenin (S276A), or a variant thereof including polynucleotides that code for conservative substitutions of alanine, such as glycine (S276G), valine (S276V), leucine (S276L) or isoleucine (S276I). In some embodiments, the polynucleotide includes SEQ ID NO:32 or a sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:32.
In another aspect, the disclosure provides compositions that include an expression cassette that includes a nucleic acid sequence encoding the human delta-catenin protein, or a variant thereof, as described herein. In some embodiments, the nucleic acid sequence in the expression cassette includes SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, or a sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30. In some embodiments, the nucleic acid sequence encodes human-delta-catenin (S276A), or a variant thereof including polynucleotides that code for conservative substitutions of alanine, such as glycine (S276G), valine (S276V), leucine (S276L) or isoleucine (S276I). In some embodiments, the polynucleotide includes SEQ ID NO:32 or a sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:32.
In another aspect, the disclosure provides compositions that include a recombinant viral vector as described below and herein. In some embodiments, the recombinant viral vector is an Adeno-associated virus as described below and herein.
Another aspect of the present disclosure provides a pharmaceutical composition including a composition according to the present disclosure and a pharmaceutically acceptable carrier and/or excipient.
Recombinant Viral Vectors
The recombinant transgenes and/or nucleic acid expression cassettes (e.g., human-delta-catenin and variants thereof) provided herein may be incorporated into recombinant viral vectors/viruses. Any suitable recombinant viral vector suitable for gene therapy is suitable for use in the compositions and methods according to the present disclosure. In some embodiments, the recombinant viral vector can include adenoviruses, Adeno-associated viruses (AAV), Herpes simplex viruses (e.g., Herpes Simplex Virus Type 1), Retroviruses, lentiviruses, alphaviruses, flaviviruses, rhabdoviruses, measles virus, Newcastle disease virus, poxviruses, or picornaviruses. In some embodiments, the recombinant viral vector includes a recombinant Adeno-Associated Viruses (AAV). In other embodiments, the recombinant viral vector comprises a recombinant lentiviral vector.
In some embodiments, the recombinant viral vectors according to the disclosure can include one or more of the following elements: an Inverted Terminal Repeat sequence (ITR), a promoter (e.g., a neuron-specific promoter), an intron, a (trans)gene (e.g., a transgene encoding human-delta-catenin, a variant thereof, a fragment thereof, an isoform thereof, or a homologue thereof), a transcription terminator, e.g., a polyadenylation signal, a flanking Inverted Terminal Repeat sequence (ITR).
In some embodiments, the promoter can include a synapsin 1 promoter, calcium/calmodulin-dependent protein kinase II promoter, tubulin alpha 1 promoter, neuron-specific enolase promoter, platelet-derived growth factor beta chain promoters, human KCC2 promoter, and the like. In one embodiment, the promoter comprises the human synapsin promoter. In another embodiment, the promoter comprises the human KCC2 promoter.
In some embodiments, the recombinant viral vector includes a constitutively active promoter. In some embodiments, the constitutively active promoter can be, for example, human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, and herpes simplex virus thymidine kinase. In some embodiments, the recombinant viral vector includes a neuro-specific promoter.
In some embodiments, the recombinant viral vector further includes a transcriptional termination signal that can be, for example, bovine growth hormone polyadenylation signal (BGHpA), Simian virus 40 polyadenylation signal (SV40pA), and a synthetic polyadenylation signal.
In some embodiments the recombinant viral vector comprises Adeno-associated virus.
Recombinant AAV genomes of the present disclosure comprise nucleic acid molecule of the present disclosure (e.g., human-delta-catenin and variants thereof) and one or more AAV ITRs flanking a nucleic acid molecule. AAV DNA in the rAAV genomes may be from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12 and AAV-13. In some embodiments, the AAV serotype used comprises AAV1, AAV8 and AAV9. In certain embodiments, the recombinant AAV comprises AAV9, and in certain embodiments, AAV-1. Production of pseudotyped rAAV are disclosed in, for example, WO 01/83692. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. (See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). It is understood that the nucleotide sequences of the genomes of various AAV serotypes are known in the art.
In some embodiments, the DNA plasmids of the present disclosure comprise rAAV genomes of the present disclosure. The DNA plasmids are transferred to cells permissible for infection with a helper virus of AAV (e.g., adenovirus, E1-deleted adenovirus or herpes virus) for assembly of the rAAV genome into infectious viral particles. Techniques to produce rAAV particles, in which an AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell, are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived and may be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12 and AAV-13. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692 which is incorporated by reference herein in its entirety. In some embodiments, the recombinant AAV vector is selected from the group consisting of AAV1, AAV8, and AAV9 and expressing a nucleotide sequence encoding the human-delta-catenin protein and variants thereof.
A method of generating a packaging cell is to create a cell line that stably expresses all the necessary components for viral (e.g., AAV) particle production. For example, in one embodiment, a plasmid (or multiple plasmids) comprising a viral rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) or by direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce rAAV genomes and/or rep and cap genes into packaging cells.
General principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various approaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat. No. 5,173,414; WO 95/13365 and corresponding U.S. Pat. No. 5,658,776; WO 95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO 97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243 (PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine 13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3:1124-1132; U.S. Pat. Nos. 5,786,211; 5,871,982; and 6,258,595. The foregoing documents are hereby incorporated by reference in their entirety herein, with particular emphasis on those sections of the documents relating to rAAV production.
The present disclosure further provides packaging cells that produce infectious recombinant viral vectors (e.g. rAAV). In one embodiment, packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293 line). In another embodiment, packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with E1 of adenovirus), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells).
In embodiments comprising a recombinant AAV, the recombinant AAV (i.e., infectious encapsidated rAAV particles) of the present disclosure comprises a rAAV genome. In exemplary embodiments, the genomes of both rAAV lack AAV rep and cap DNA, that is, there is no AAV rep or cap DNA between the ITRs of the genomes. Examples of rAAV that may be constructed to comprise the nucleic acid molecules of the invention are set out in International Patent Application No. PCT/US2012/047999 (WO 2013/016352) incorporated by reference herein in its entirety.
The recombinant viral vectors (e.g., rAAV) may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying recombinant viral vectors from helper virus are known in the art and include methods disclosed in, for example, Clark et al., Hum. Gene Ther., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002); U.S. Pat. No. 6,566,118 and WO 98/09657.
Pharmaceutical Compositions
In another aspect, the present disclosure provides pharmaceutical compositions comprising a recombinant viral vector comprising the human-delta-catenin transgene and/or nucleic acid expression cassettes comprising the human-delta-catenin protein as described herein. Other pharmaceutical compositions contemplated in the present disclosure include those comprising GSK3β inhibitors. In some embodiments, compositions of the present disclosure include a recombinant viral vector and/or a pharmaceutically acceptable carrier and/or excipient. In other embodiments, compositions of the present disclosure include a GSK3β inhibitor as provided herein and/or a pharmaceutically acceptable carrier and/or excipient.
Pharmaceutically acceptable compositions are prepared in view of approvals for a regulatory agency or other agency prepared in accordance with generally recognized pharmacopeia for use in animals and in humans. The compounds can be formulated into any suitable pharmaceutical preparations for any of injectable, oral or topical administration such as solutions, suspensions, powders, or sustained release formulations. Typically, the compounds are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition, 1985, 126. The formulation should suit the mode of administration.
In one example, pharmaceutical preparation can be in liquid form, for example, solutions, syrups or suspensions. If provided in liquid form, the pharmaceutical preparations can be provided as a concentrated preparation to be diluted to a therapeutically effective concentration before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). In another example, pharmaceutical preparations can be presented in lyophilized form for reconstitution with water or other suitable vehicle before use.
Pharmaceutical compositions can include carriers such as a diluent, adjuvant, excipient, or vehicle with which the composition (e.g. GSK3beta inhibitor or recombinant transgene, recombinant viral vector, or recombinant AAV vector) are administered. Acceptable carriers, diluents and adjuvants are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter ions such as sodium; and/or nonionic surfactants such as Tween, pluronics or polyethylene glycol (PEG).
Pharmaceutically acceptable carriers used in parenteral preparations include, for example, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances. Examples of aqueous vehicles include Sodium Chloride Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to parenteral preparations packaged in multiple-dose containers, which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcelluose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEENs 80). A sequestering or chelating agent of metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment. Further examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Preparations for intraprostatic administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, sterile emulsions. The solutions can be either aqueous or nonaqueous.
Titers of recombinant viral vectors to be administered according to the methods of the present disclosure will vary depending, for example, on the particular recombinant viral vector, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art. Titers of recombinant viral vector may range from about 1×106, about 1×107, about 1×108, about 1×109, about 1×1010, about 1×1011, about 1×1012, about 1×1013, about 1×1014, or to about 1×1015 or more DNase resistant particles (DRP) per ml. Dosages may also be expressed in units of viral genomes (vg).
Methods of transducing a target cell with a recombinant viral vector according to the present disclosure, in vivo or in vitro, are contemplated by the present disclosure. The in vivo methods comprise the step of administering an effective dose, or effective multiple doses, of a composition comprising a recombinant viral vector of the present disclosure to an animal (including a human being) in need thereof. If the dose is administered prior to development of a disorder/disease, the administration is prophylactic. If the dose is administered after the development of a disorder/disease, the administration is therapeutic. In embodiments of the present disclosure, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder/disease state being treated, that slows or prevents progression to a disorder/disease state, that slows or prevents progression of a disorder/disease state, that diminishes the extent of disease, that results in remission (partial or total) of disease, and/or that prolongs survival. An example of a disease contemplated for prevention or treatment with methods of the present disclosure is pain and/itch.
Combination therapies are also contemplated by the present disclosure. Combination as used herein includes both simultaneous treatment and sequential treatments. Combinations of methods of the present disclosure with standard medical treatments are specifically contemplated, as are combinations with novel therapies. Combination therapies may include the administration of a recombinant viral vector according to the present disclosure along with a GSK3β inhibitor. Alternatively, a recombinant viral vector and/or GSK3β inhibitor according to the present disclosure may be administered with another therapeutic compound such as, but not limited to, anti-analgesics (e.g., NSAIDS, corticosteroids, acetaminophen, narcotic analgesics (e.g., opioids), muscle relaxants, anti-anxiety drugs, antidepressants, anticonvulsants, and the like) and/or anti-pruritics, such as corticosteroids (e.g., hydrocortisone), counterirritants (e.g., mint oil, menthol, camphor), antihistamines, local anesthetics (e.g., lidocaine, pramoxine, benzocaine) and the like and/or GSK3 inhibitors as provided herein.). In some embodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different recombinant viral vectors, e.g., rAAVs, each having one or more different transgenes.
Administration of an effective dose of the compositions may be by routes standard in the art including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. Route(s) of administration and serotype(s) of viral (e.g., AAV) components of the recombinant viral vector (e.g., rAAV, and in particular, the AAV ITRs and capsid protein) of the present disclosure may be chosen and/or matched by those skilled in the art taking into account the disease state being treated and the target cells/tissue(s) that are to express the human-delta-catenin protein.
The present disclosure further provides for local administration and systemic administration of an effective dose of a composition (e.g. recombinant viral vector and/or a GSK3beta inhibitor) and compositions of the present disclosure including combination therapy as provided herein. For example, systemic administration is administration into the circulatory system so that the entire body is affected. Systemic administration includes enteral administration such as absorption through the gastrointestinal tract and parenteral administration through injection, infusion or implantation.
In particular, actual administration of a composition according to the present disclosure may be accomplished by using any physical method that will transport the composition into the target tissue of the subject. Administration according to the present disclosure includes, but is not limited to, injection into the ventricles, cisterna magna, spinal theca, muscle, the bloodstream and/or directly into the brain. Simply resuspending the composition in phosphate buffered saline (PBS) has been demonstrated to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known restrictions on the carriers or other components that can be co-administered with the composition. In cases where the composition comprises a recombinant viral vector (e.g., rAAV), compositions that degrade DNA should be avoided in the normal manner with rAAV. In those cases where the recombinant viral vector comprises rAAV, the capsid proteins of a rAAV may be modified so that the rAAV is targeted to a particular target tissue of interest such as muscle. See, for example, WO 02/053703, the disclosure of which is incorporated by reference herein. Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations to be delivered to the subject by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been previously developed and can be used in the practice of the invention. The recombinant viral vector can be used with any pharmaceutically acceptable carrier and/or excipient for ease of administration and handling.
In those embodiments where the composition comprises a recombinant viral vector (including a recombinant AAV vector), the dose to be administered in methods disclosed herein will vary depending, for example, on the particular recombinant viral vector, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art. Titers of each recombinant viral vector (e.g., rAAV) administered may range from about 1×106, about 1×107, about 1×108, about 1×109, about 1×1010, about 1×1011, about 1×1012, about 1×1013, about 1×1014, or to about 1×1015 or more DNase resistant particles (DRP) per ml. Dosages may also be expressed in units of viral genomes (vg) (i.e., 1×107 vg, 1×108 vg, 1×109 vg, 1×1010 vg, 1×1011 vg, 1×1012 vg, 1×1013 vg, 1×1014 vg, 1×1015 respectively). Dosages may also be expressed in units of viral genomes (vg) per kilogram (kg) of bodyweight (i.e., 1×1010 vg/kg, 1×1011 vg/kg, 1×1012 vg/kg, 1×1013 vg/kg, 1×1014 vg/kg, 1×1015 vg/kg respectively). Methods for determining titer of viral vectors such as AAV are described in Clark et al., Hum. Gene Ther., 10: 1031-1039 (1999).
For purposes of intramuscular injection, solutions in an adjuvant such as sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions. Such aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose. Solutions of a composition according to the present disclosure as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxpropylcellulose. A dispersion of composition according to the present disclosure can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain one or more preservatives of chemical stabilizers to prevent the growth of microorganisms and prolong product life. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin. Sterile aqueous media is typically employed.
In some embodiments, recombinant viral vector, e.g., rAAV, compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present (e.g., ˜1013 GC/ml or more). Methods for reducing aggregation of rAAVs are well known in the art and, include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright F R, et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference).
rAAV compositions disclosed herein may also be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.
The pharmaceutical carriers, diluents or excipients suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy use in a syringe is possible. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating actions of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.
In some embodiments, sterile injectable solutions are prepared by incorporating the composition(s) according to the present disclosure in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.
Transduction with a recombinant viral vector may also be carried out in vitro. In one embodiment, desired target cells are removed from the subject, transduced with recombinant viral vector and reintroduced into the subject. Alternatively, syngeneic or xenogeneic target cells can be used where those cells will not generate an inappropriate immune response in the subject.
Suitable methods for the transduction and reintroduction of transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro by combining the recombinant viral vector with target cells, e.g., in appropriate media, and screening for those cells harboring the DNA of interest using conventional techniques such as Southern blots and/or PCR, or by using selectable markers. Transduced cells can then be formulated into pharmaceutical compositions, and the composition introduced into the subject by various techniques, such as by intramuscular, intravenous, subcutaneous and intraperitoneal injection, or by injection into smooth and cardiac muscle, using e.g., a catheter.
Transduction of cells with recombinant viral vector(s) of the present disclosure can result in sustained expression of human-delta-catenin. The present disclosure thus provides methods of administering/delivering a recombinant viral vector which expresses human-delta-catenin to a subject, preferably a human being. These methods include transducing tissues (including, but not limited to, tissues such as muscle, organs such as liver and brain, and glands such as salivary glands) with one or more recombinant viral vector of the present disclosure. Transduction may be carried out with gene cassettes comprising tissue specific control elements as described herein. The term “transduction” is used to refer to the administration/delivery of the human-delta-catenin gene to a recipient cell either in vivo or in vitro, via a replication-deficient recombinant viral vector of the present disclosure thereby resulting in expression of human-delta-catenin by the recipient cell. Thus, the present disclosure provides methods of administering an effective dose (or doses, administered essentially simultaneously or doses given at intervals) of a recombinant viral vector that encodes human-delta-catenin to a subject in need thereof.
Targeting CNS Tissue
Methods for delivering a transgene to central nervous system (CNS) tissue in a subject are provided herein. A method for delivering a transgene to CNS tissue in a subject may comprise administering a rAAV by a single route or by multiple routes. For example, delivering a transgene to CNS tissue in a subject may comprise administering to the subject, by intravenous administration, an effective amount of a rAAV that crosses the blood-brain-barrier. Delivering a transgene to CNS tissue in a subject may comprise administering to the subject an effective amount of a rAAV by intrathecal administration or intracerebral administration, e.g., by intraventricular injection. A method for delivering a transgene to CNS tissue in a subject may comprise co-administering of an effective amount of a rAAV by two different administration routes, e.g., by intrathecal administration and by intracerebral to administration. Co-administration may be performed at approximately the same time, or different times.
The CNS tissue to be targeted may be selected from cortex, hippocampus, thalamus, hypothalamus, cerebellum, brain stem, cervical spinal cord, thoracic spinal cord, and lumbar spinal cord, for example. The administration route for targeting CNS tissue typically depends on the AAV serotype. For example, in certain instances where the AAV serotype is selected from AAV1, AAV6, AAV6.2, AAV7, AAV8, AAV9, rh.10, rh.39, rh.43 and CSp3, the administration route may be intravascular injection. In some instances, for example where the AAV serotype is selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, rh.10, rh.39, rh.43 and CSp3, the administration route may be intrathecal and/or intracerebral injection. The disclosure describes the use of one or more serotypes, including AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV 11, AAV12, rh.10, rh.39, rh.43 and CSp3 or AAV13
Intravascular Administration
As used herein the term “intravascular administration” refers to the administration of an agent, e.g., a composition comprising a recombinant viral vector, e.g., rAAV, into the vasculature of a subject, including the venous and arterial circulatory systems of the subject. rAAVs that cross the blood-brain-barrier may be delivered by intravascular administration for targeting CNS tissue. In some cases, intravascular (e.g., intravenous) administration facilitates the use of larger volumes than other forms of administration (e.g., intrathecal, intracerebral). Thus, large doses of rAAVs (e.g., up to 1015 GC/subject) can be delivered at one time by intravascular (e.g., intravenous) administration. Methods for intravascular administration are well known in the art and include for example, use of a hypodermic needle, peripheral cannula, central venous line, etc.
Intrathecal and/or Intracerebral Administration
As used herein the term “intrathecal administration” refers to the administration of an agent, e.g., a composition comprising a rAAV, into the spinal canal. For example, intrathecal administration may comprise injection in the cervical region of the spinal canal, in the thoracic region of the spinal canal, or in the lumbar region of the spinal canal. Typically, intrathecal administration is performed by injecting an agent, e.g., a composition comprising a rAAV, into the subarachnoid cavity (subarachnoid space) of the spinal canal, which is the region between the arachnoid membrane and pia mater of the spinal canal. The subarchnoid to space is occupied by spongy tissue consisting of trabecula (delicate connective tissue filaments that extend from the arachnoid mater and blend into the pia mater) and intercommunicating channels in which the cerebrospinal fluid is contained. In some embodiments, intrathecal administration is not administration into the spinal vasculature.
As used herein, the term “intracerebral administration” refers to administration of an agent into and/or around the brain. Intracerebral administration includes, but is not limited to, administration of an agent into the cerebrum, medulla, pons, cerebellum, intracranial cavity, and meninges surrounding the brain. Intracerebral administration may include administration into the dura mater, arachnoid mater, and pia mater of the brain. Intracerebral administration may include, in some embodiments, administration of an agent into the cerebrospinal fluid (CSF) of the subarachnoid space surrounding the brain. Intracerebral administration may include, in some embodiments, administration of an agent into ventricles of the brain, e.g., the right lateral ventricle, the left lateral ventricle, the third ventricle, the fourth ventricle. In some embodiments, intracerebral administration is not administration into the brain vasculature.
Intracerebral administration may involve direct injection into and/or around the brain. In some embodiments, intracerebral administration involves injection using stereotaxic procedures. Stereotaxic procedures are well known in the art and typically involve the use of a computer and a 3-dimensional scanning device that are used together to guide injection to a particular intracerebral region, e.g., a ventricular region. Micro-injection pumps (e.g., from World Precision Instruments) may also be used. In some embodiments, a microinjection pump is used to deliver a composition comprising a recombinant viral vector, e.g., rAAV. In some embodiments, the infusion rate of the composition is in a range of 1 μl/minute to 100 μl/minute. As will be appreciated by the skilled artisan, infusion rates will depend on a variety of factors, including, for example, species of the subject, age of the subject, weight/size of the subject, serotype of the AAV, dosage required, intracerebral region targeted, etc. Thus, other infusion rates may be deemed by a skilled artisan to be appropriate in certain circumstances.
The disclosure also provides that delivery vehicles, such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present invention into suitable host cells, e.g., neurons. The recombinant viral vector-delivered (e.g., rAAV-delivered) transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the rAAV constructs disclosed herein. The formation and use of liposomes is generally known to those of skill in the art. Liposomes were developed with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).
Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed.
Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 Angstrom, containing an aqueous solution in the core.
Alternatively, nanocapsule formulations of the rAAV may be used. Nanocapsules can generally entrap substances in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 m) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.
In addition to the methods of delivery described above, the following techniques are also contemplated as alternative methods of delivering the rAAV compositions to a host. Sonophoresis (ie., ultrasound) has been used and described in U.S. Pat. No. 5,656,016 as a device for enhancing the rate and efficacy of drug permeation into and through the circulatory system. Other drug delivery alternatives contemplated are intraosseous injection (U.S. Pat. No. 5,779,708), microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) and feedback-controlled delivery (U.S. Pat. No. 5,697,899).
Methods of Use
Another aspect of the disclosure provides methods of treating pain and/or itch in a subject in need thereof, the method comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a transgene, nucleic acid cassette, a recombinant virus genetically modified to express human-delta-catenin protein, or a variant thereof, and any pharmaceutical compositions thereof such that the pain and/or itch is treated in the subject.
In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of at least one additional compound. In some embodiments, the compound is selected from the group consisting of a GSK3 inhibitor, anti-analgesics (e.g., NSAIDS, corticosteroids, acetaminophen, narcotic analgesics (e.g., opioids), muscle relaxants, anti-anxiety drugs, antidepressants, anticonvulsants, and the like) and/or anti-pruritics, such as corticosteroids (e.g., hydrocortisone), counterirritants (e.g., mint oil, menthol, camphor), antihistamines, local anesthetics (e.g., lidocaine, pramoxine, benzocaine) and the like.
In another aspect, the disclosure provides methods related to the transgenes, expression cassettes, recombinant viral vectors, recombinant AAV vectors, compositions and pharmaceutical compositions as described in detail above and herein, for example, in the Compositions, Recombinant Viral Vectors and Pharmaceutical Composition sections (collectively “composition” or “compositions”).
In some embodiments, methods of treating pain in a subject in need thereof are provided, including administering a therapeutically effective amount of a composition as described above and herein such that the pain is treated in the subject. In some embodiments, the pain is neuropathic pain or pathological pain. In some embodiments, methods of treating itch in a subject are provided, including administering to the subject a therapeutically effective amount of a composition described above and herein such that the itch is treated in the subject. In some embodiments, the itch is neuropathic or pathological itch.
In some embodiments, methods of increasing KCC2 mRNA levels in a subject are provided, including administering to the subject a therapeutically effective amount of a composition as described above and herein so that the KCC2 mRNA levels are increased in the subject compared to a pre-treatment baseline. In some embodiments, KCC2 mRNA levels are increased at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 125%, or at least about 150%, or at least about 175%, about 200%, or more than 200% compared to pre-treatment baseline, and in some embodiments, KCC2mRNA levels are increased at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 125%, or at least about 150% compared to pre-treatment baseline.
In some embodiments, methods of reducing intracellular chloride ion levels ([Cl-]i) in a central nervous system cell in a subject are provided, including administering to the subject a therapeutically effective amount of a composition as described above and herein so that the [Cl—]i in a central nervous system cell is reduced in the subject compared to a pre-treatment baseline. In some embodiments, [Cl—]i levels are reduced at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to pre-treatment baseline.
In some embodiments, methods of increasing chloride ion efflux in a central nervous system cell in a subject are provided, including administering to the subject a therapeutically effective amount of a composition as described above and herein so that the chloride ion efflux is increased in the subject. In some embodiments, chloride ion efflux levels are increased at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 110%, or at least about 120%, or at least about 130%, or at least about 140%, or at least about 150%, or at least about 160%, or at least about 170%, or at least about 180%, or at least about 190%, about 200%, or more than 200% compared to pre-treatment baseline.
In some embodiments, methods of increasing synaptophysin expression in a central nervous system cell in a subject are provided, including administering to the subject a therapeutically effective amount of a composition as described above and herein so that the synaptophysin expression is increased in the subject as compared to a pre-treatment baseline. In some embodiments, synaptophysin expression is increased at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 110%, or at least about 120%, or at least about 130%, or at least about 140%, or at least about 150%, or at least about 160%, or at least about 170%, or at least about 180%, or at least about 190%, about 200%, or more than 200% compared to pre-treatment baseline, and in some embodiments, synaptophysin expression is increased at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 125%, or at least about 150% compared to pre-treatment baseline.
In some embodiments, methods of increasing KCC2 expression in a subject are provided, including administering to the subject a therapeutically effective amount of a composition as described above and herein so that KCC2 expression is increased in the subject compared to pre-treatment baseline. In some embodiments, KCC2 expression is increased at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 125%, or at least about 150%, or at least about 175%, about 200%, or more than 200% compared to pre-treatment baseline, and in some embodiments KCC2mRNA levels are increased at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 125%, or at least about 150% compared to pre-treatment baseline.
In some embodiments of the methods described above and herein, the methods further include administering to the subject a therapeutically effective amount of at least one additional compound, which compound, in some embodiments, includes a GSK3β inhibitor, an anti-analgesic, a muscle relaxant, an anti-anxiety drug, an antidepressant, an anticonvulsant, and combinations thereof. In some embodiments, the at least one additional compound includes a GSK3β inhibitor a corticosteroid, a counterirritant, an antihistamine, and a local anesthetic and combinations thereof.
In some embodiments, the GSK3β inhibitor is selected from the group consisting of CHIR-99021 (CT99021) HCl, SB-216763, CHIR-98014, TWS119, Tideglusib, SB-415286, BIO (6-bromoindirubin-3′-oxime), kenpaullone, CHIR-99021 (CT99021), AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, IM-12, BIO-acetoxime, Indirubin, 5-Bromoindole, 2-D08, Bilinin, 1-Azakenpaullone, lithium chloride, lithium carbonate, lithium citrate, lithium orotate, lithium bromide, lithium fluoride, lithium iodide, lithium acetate, lithium hydroxide, lithium aluminum hydride, lithium perchlorate, lithium nitrate, lithium diisopropylamide, lithium borohydride, lithium oxide, lithium sulfate, lithium hexafluorophosphate, lithium tetroxide, lithium sulfide, lithium hydride, lithium amide, lithium lactate, lithium tetrafluoroborate, lithium dimethylamide, lithium phosphate, lithium peroxide, lithium manganese oxide, lithium methoxide, lithium metaborate, lithium stearate, or another lithium salt that comprises cationic lithium and any combinations thereof. In some embodiments, the GSK3β inhibitor is selected from the group consisting of kenpaullone (9-bromo-7,12-dihydro-indolo [3,2-d] [1]benzazepin-6(5H)-one), NSC180515 (2-Acetyl-2,3,4,5-tetrahydrooxonine-6,9-dione), NSC79456 (n-(2,4-Dimethylphenyl)-2-hydroxy-3-nitrobenzamide), or NSC33006 (N-[4-(1,3-benzothiazol-2-yl)phenyl]acetamide), and in some embodiments, the GSK3β inhibitor is kenpaullone (9-bromo-7,12-dihydro-indolo [3,2-d][1]benzazepin-6(5H)-one).
Another aspect of the disclosure is based on the findings by the inventor that renormalizing inhibitory neurotransmission can be achieved by GSK3β inhibition.
Accordingly, another aspect of the present disclosure provides a method of treating pain and/or itch in a subject in need thereof, the method comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a GSK3β inhibitor, or a pharmaceutical composition comprising a GSK3β inhibitor and a pharmaceutically acceptable carrier and/or excipient, such that the pain and/or itch is treated in the subject.
As used herein, the term “GSK3β inhibitor” refers to any compound/molecule (e.g., antibodies, small molecules, oligonucleotides, siRNAs, miRNAs, etc.) that is able to inhibit or reduce the function of the GSK3β protein, or inhibit or reduce the expression of the GSK3 gene. Suitable inhibitors may include, but are not limited to, CHIR-99021 (CT99021) HCl, SB-216763, CHIR-98014, TWS119, Tideglusib, SB-415286, BIO (6-bromoindirubin-3′-oxime), kenpaullone, CHIR-99021 (CT99021), AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, IM-12, BIO-acetoxime, Indirubin, 5-Bromoindole, 2-D08, Bilinin, 1-Azakenpaullone, lithium chloride, lithium carbonate, lithium citrate, lithium orotate, lithium bromide, lithium fluoride, lithium iodide, lithium acetate, lithium hydroxide, lithium aluminum hydride, lithium perchlorate, lithium nitrate, lithium diisopropylamide, lithium borohydride, lithium oxide, lithium sulfate, lithium hexafluorophosphate, lithium tetroxide, lithium sulfide, lithium hydride, lithium amide, lithium lactate, lithium tetrafluoroborate, lithium dimethylamide, lithium phosphate, lithium peroxide, lithium manganese oxide, lithium methoxide, lithium metaborate, lithium stearate, or another lithium salt that comprises cationic lithium and any pharmaceutical compositions thereof.
In some embodiments, the disclosure provides methods of treating pain and/or treating itch in a subject in need thereof, including administering a therapeutically effective amount of a GSK3β inhibitor, or a pharmaceutical composition including a GSK3β inhibitor and a pharmaceutically acceptable carrier and/or excipient, such that the pain is treated in the subject. In some embodiments, the pain and/or itch is neuropathic or pathological in nature.
In some embodiments, methods of increasing KCC2 mRNA levels in a subject are provided, including administering to the subject a therapeutically effective amount of a GSK3β inhibitor, or a pharmaceutical composition including a GSK3β inhibitor and a pharmaceutically acceptable carrier and/or excipient, so that the KCC2 mRNA levels are increased in the subject. In some embodiments, KCC2 mRNA levels are increased at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 125%, or at least about 150%, or at least about 175%, about 200%, or more than 200% compared to pre-treatment baseline, and in some embodiments, KCC2mRNA levels are increased at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 125%, or at least about 150% compared to pre-treatment baseline.
In some embodiments, methods of reducing intracellular chloride ion levels ([Cl-]i) in a central nervous system cell in a subject are provided, including administering to the subject a therapeutically effective amount of a GSK3β inhibitor, or a pharmaceutical composition including a GSK3β inhibitor and a pharmaceutically acceptable carrier and/or excipient, so that the [Cl—]i in a central nervous system cell is reduced in the subject. In some embodiments, [Cl—]i levels are reduced at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to pre-treatment baseline.
In some embodiments, methods of increasing chloride ion efflux in a central nervous system cell in a subject are provided, including administering to the subject a therapeutically effective amount of a GSK3β inhibitor, or a pharmaceutical composition including a GSK3β inhibitor and a pharmaceutically acceptable carrier and/or excipient, so that the chloride ion efflux is increased in the subject. In some embodiments, chloride ion efflux levels are increased at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 110%, or at least about 120%, or at least about 130%, or at least about 140%, or at least about 150%, or at least about 160%, or at least about 170%, or at least about 180%, or at least about 190%, about 200%, or more than 200% compared to pre-treatment baseline.
In some embodiments, methods of increasing synaptophysin expression in a central nervous system cell in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a GSK3β inhibitor, or a pharmaceutical composition comprising a GSK3β inhibitor and a pharmaceutically acceptable carrier and/or excipient, such that the synaptophysin expression is increased in the subject. In some embodiments, synaptophysin expression is increased at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 110%, or at least about 120%, or at least about 130%, or at least about 140%, or at least about 150%, or at least about 160%, or at least about 170%, or at least about 180%, or at least about 190%, about 200%, or more than 200% compared to pre-treatment baseline, and in some embodiments, synaptophysin expression is increased at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 125%, or at least about 150% compared to pre-treatment baseline.
In some embodiments, methods of increasing KCC2 expression in a subject are provided, including administering to the subject a therapeutically effective amount of a GSK3β inhibitor, or a pharmaceutical composition including a GSK3β inhibitor and a pharmaceutically acceptable carrier and/or excipient, so that KCC2 expression is increased in the subject. In some embodiments, KCC2 expression is increased at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 125%, or at least about 150%, or at least about 175%, about 200%, or more than 200% compared to pre-treatment baseline, and in some embodiments KCC2mRNA levels are increased at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 125%, or at least about 150% compared to pre-treatment baseline.
In some embodiments of the methods described above, the methods further include administering to the subject a therapeutically effective amount of at least one additional compound, which compound, in some embodiments, includes compositions including a transgene, an expression cassette, or a recombinant viral vector as described above and herein (each, in some embodiments, pharmaceutical compositions that include a pharmaceutically acceptable carrier and/or excipient), an anti-analgesic (e.g., NSAIDS, corticosteroids, acetaminophen), narcotic analgesics (e.g., opioids), a muscle relaxant, an anti-anxiety drug, an antidepressant, an anticonvulsant, and combinations thereof. In some embodiments, the additional compound can include compositions including a transgene, an expression cassette, or a recombinant viral vector as described above and herein (each, in some embodiments, pharmaceutical compositions that include a pharmaceutically acceptable carrier and/or excipient), and/or anti-pruritics, such as a corticosteroid (e.g., hydrocortisone), a counterirritant (e.g., mint oil, menthol, camphor), an antihistamine, and a local anesthetic (e.g., lidocaine, pramoxine, benzocaine) and combinations thereof.
In the methods described in all relevant aspects and embodiments of the disclosure, the central nervous system cell is a neuron.
In the methods described in all relevant aspects and embodiments of the disclosure, the composition can be administered by any administration route, including intrathecally, intra-cerebroventricularly, intra-cerebrally, perispinally, intra-spinally, intravenously and others.
In embodiments of this aspect related to methods of treating pain and/or treating itch in a subject in need thereof, including administering a therapeutically effective amount of a GSK3β inhibitor, or a pharmaceutical composition including a GSK3β inhibitor, the GSK3β inhibitor includes one or more of CHIR-99021 (CT99021) HCl, SB-216763, CHIR-98014, TWS119, Tideglusib, SB-415286, BIO (6-bromoindirubin-3′-oxime), kenpaullone, CHIR-99021 (CT99021), AZD2858, AZD1080, AR-A014418, TDZD-8, LY2090314, IM-12, BIO-acetoxime, Indirubin, 5-Bromoindole, 2-D08, Bilinin, 1-Azakenpaullone, lithium chloride, lithium carbonate, lithium citrate, lithium orotate, lithium bromide, lithium fluoride, lithium iodide, lithium acetate, lithium hydroxide, lithium aluminum hydride, lithium perchlorate, lithium nitrate, lithium diisopropylamide, lithium borohydride, lithium oxide, lithium sulfate, lithium hexafluorophosphate, lithium tetroxide, lithium sulfide, lithium hydride, lithium amide, lithium lactate, lithium tetrafluoroborate, lithium dimethylamide, lithium phosphate, lithium peroxide, lithium manganese oxide, lithium methoxide, lithium metaborate, lithium stearate, or another lithium salt that comprises cationic lithium and any combinations thereof.
In some embodiments, the GSK3β inhibitor includes one or more of kenpaullone (9-bromo-7,12-dihydro-indolo [3,2-d] [1]benzazepin-6(5H)-one), NSC180515 (2-Acetyl-2,3,4,5-tetrahydrooxonine-6,9-dione), NSC79456 (n-(2,4-Dimethylphenyl)-2-hydroxy-3-nitrobenzamide), or NSC33006 (N-[4-(1,3-benzothiazol-2-yl)phenyl]acetamide), and in some embodiments, the GSK3β inhibitor is kenpaullone (9-bromo-7,12-dihydro-indolo [3,2-d][1]benzazepin-6(5H)-one).
In some embodiments, the methods further comprising administering to the subject a therapeutically effective amount of at least one additional compound, which, in some embodiments, includes one or more compositions (e.g., transgenes, expression cassettes, recombinant viral vectors, recombinant AAV vectors, compositions and pharmaceutical compositions as described in detail above and herein, for example, in the Compositions, Recombinant Viral Vectors and Pharmaceutical Composition sections), an anti-analgesic, a muscle relaxant, an anti-anxiety drug, an antidepressant, an anticonvulsant, and combinations thereof. In some embodiments, the additional compound includes one or more compositions (as above), a corticosteroid, a counterirritant, an antihistamine, and a local anesthetic and combinations thereof.
In some embodiments, the central nervous system cell is selected from the group consisting of neurons, oligodendrocytes, astrocytes, brain parenchyma cells, and Purkinje cells, and in some embodiments, the central nervous system cell is a neuron.
In some embodiments of all aspects of the disclosure, compositions (in its broadest sense, e.g., one or more of transgenes, expression cassettes, recombinant viral vectors, recombinant AAV vectors, compositions and pharmaceutical compositions as described in detail above and herein, for example, in the Compositions, Recombinant Viral Vectors and Pharmaceutical Composition sections, GSK3β inhibitors, anti-analgesic, a muscle relaxant, an anti-anxiety drug, an antidepressant, an anticonvulsant, corticosteroid, a counterirritant, an antihistamine, and a local anesthetic as described herein) are administered intrathecally, intra-cerebroventricularly, intra-cerebrally, perispinally, intra-spinally, intravascularly, intravenously, orally, enterally, rectally, pulmonarily, via inhalation, nasally, topically, transdermally, buccally, sublingually, intravesically, intravitreally, intraperitoneally, vaginally, intrasynovially, intracutaneously, intraarticularly, intraarterially, parenterally, subcutaneously, intrastemally, intralesionally, intramuscularly, intravenously, intradermally, transmucosally, or sublingually.
In some embodiments, the composition is administered intrathecally, intra-cerebroventricularly, intra-cerebrally, perispinally, intra-spinally, and in some embodiments, the composition is administered intrathecally.
In an aspect, the disclosure provides a method for delivering a transgene to central nervous system tissue in a subject, the method comprising administering an effective amount of a rAAV comprising a promoter operably linked with a transgene to central nervous system (CNS) tissue by intrathecal administration, wherein the rAAV infects cells of the CNS of the subject, wherein the transgene encodes a polypeptide of any one of SEQ ID NO:21, 23, 25, 27, 29, 31 or 33 (or a sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 21, 23, 25, 27, 29, 31 or 33), wherein the polypeptide reduces pathologic pain or itch.
In some embodiments, the intrathecal administration is in the lumbar region of the subject; in some embodiments the intrathecal administration is in the cervical region of the subject, and in some embodiments the intrathecal administration is in the thoracic region of the subject.
In some embodiments, the cells of central nervous system tissue include oligodendrocytes, astrocytes, neurons, brain parenchyma cells, and/or Purkinje cells, and in some embodiments, the cells of central nervous system tissue are neurons.
As described in the Examples herein, an unbiased screen was conducted of cell growth-regulating compounds with the insight that a sizable fraction of them function by interfering with epigenetic and transcriptional machinery. Since mature neurons do not divide, these compounds were attractive candidates to upregulate gene expression of KCC2 and thereby lower neuronal chloride levels. The inventor identified such compounds by rigorous iterations of primary and secondary screening. In particular, the inventor identified kenpaullone (KP), a glycogen synthase kinase-3 (GSK3)/cyclin-dependent kinase (CDK) inhibitor (Schultz et al., 1999, J Med Chem 42:2909-2919; Zaharevitz et al., 1999, Cancer research 59:2566-2569). KP functions as an analgesic and antipruritic in preclinical mouse models. The data described in the Examples herein suggest that a cellular mechanism of action of KP in neurons is based on its GSK3ß-inhibitory function which causes increased KCC2 gene expression, which in turn relies on nuclear transfer of the neuronal catenin, δ-catenin (referred to herein as δ-cat, Δ-cat, or delta-cat) (Arikkath et al., 2009, J Neurosci 29:5435-5442; Turner et al., 2015, Nature 520:51-56). In the nucleus, the inventor found that δ-cat enhances KCC2 gene expression via Kaiso transcription factors (Rodova et al., 2004, Mol Cell Biol 24:7188-7196). Increased KCC2 gene expression led to increased KCC2 chloride extrusion in neurons. The inventor also documented KCC2 expression enhancement in the spinal cord dorsal horn (SCDH) of mice with nerve injury, including more negative, thus electrically more stable GABA-evoked chloride reversal potential.
The following Examples are provided by way of illustration and not by way of limitation.
Materials and Methods
Screening in Primary Cortical Neurons from KCC2-LUC Transgenic Mice
Transgenic mice that express red-shifted luciferase (LUC) under the control of the KCC2 promoter (−2052/+476, described in (Yeo et al., 2009, J Neurosci 29:14652-14662)), inserted into the Rosa26 locus, were previously described (Liedtke et al., 2013, Small 9:1066-1075; Yeo et al., 2013, Proc Natl Acad Sci USA 110:4315-4320). Primary cortical neuronal cultures were generated from newborn (p0) mice of this line as previously described (Liedtke et al., 2013, Small 9:1066-1075; Yeo et al., 2013, Proc Natl Acad Sci USA 110:4315-4320).
Cytosine arabinoside (2.5 m) was added to cultures on the second day after seeding [2 d in vitro (DIV)] to inhibit the proliferation of non-neuronal cells. Cell suspension was plated at a density of 1×106 cells/ml onto 24-well tissue-culture dishes coated with poly-d-lysine. Cortical neuronal cultures prepared by this method yielded a majority population of neuronal cells, with negligible glia contamination, as evidenced by the absence of GFAP by Western blotting.
After a week in culture, neurons were treated with compounds (100 nM for 48 h). LUC activity was then determined for each compound. Culture supernatant was removed and cells were lysed with 150 μl lysis buffer (Targeting Systems CA, USA cat. CLR1). LUC activity was measured with a RedLuciferase Assay kit (Targeting Systems cat. FLAR) according to the manufacturer's instructions. A Veritas microplate luminometer was used to measure luminescence; for each treatment triplicates of 40 μl cell lysates (from each 24-well tissue-culture dish) were used and 25 μl substrate was injected per well. To evaluate the quality of screening methodology, Z′ factor (Zhang et al., 1999, Journal of biomolecular screening 4:67-73) was ascertained using 0.5% (v/v) DMSO as negative control and Trichostatin A as positive control. Relative Light units (RLU) for each treatment was derived from Light Units (compound treatment)/vehicle treatment (0.5% DMSO).
The primary screen encompassed three levels of LUC measurements. The first level of screening yielded a total of 137 compounds with cut-off RLU>125% LUC activity. These 137 compounds were subject to second round of screening, which was carried out in duplicate independent assays to yield the top 103 compounds sorted for highest RLU. The 103 compounds were then subjected to a third round of screening carried out in duplicate independent assays to yield the top 40 compounds which were again ranked. The best 22 of these 40 compounds (ranked for highest RLU activity) were subjected to secondary screening: Cultured neurons were treated with the 22 compounds; RT-qPCR and Clomeleon imaging methodologies (Yeo et al., 2009, J Neurosci 29:14652-14662) were used to determine effects of compounds on KCC2 mRNA expression and [Cl—]i, respectively. Each compound was ranked based on composite scores from primary and secondary screening.
Compound Libraries
NCI compound libraries Natural Products II and Mechanistic Diversity Set II were obtained from the National Cancer Institute. See also: https://dtp.cancer.gov/organization/dscb/obtaining/available_plates.htm.
Human Neuronal Cultures
The neuron-enriched cultures were established from fetal cortical specimens at 15-20 weeks of gestation. The protocols for tissue processing complied with all federal and institutional guidelines. The cultures were plated on PEI (polyethyleneimine solution) substrate and maintained in Neurobasal media supplemented with B27 as previously described (Pelsman et al., 2003, International journal of developmental neuroscience 21:117-124; Yeo et al., 2013, Proc Natl Acad Sci USA 110:4315-4320).
For RT-qPCR, the cell pellets were collected after treatments with vehicle (0.1% DMSO) or KP (50, 100, and 400 nM) for 48 h starting at day 6 in vitro.
For immunostaining, the cultures were treated for 2-4 days with vehicle or 400 nM KP, fixed with 4% PFA at day 10, and processed for double-staining with anti-KCC2 and anti-synaptophysin or anti-NeuN (see Table 1).
The 0.34 micrometer confocal slices through entire cell layers at different optical fields (n=17-28 for each group) of the fixated cultures were acquired using a Zeiss LSM700 confocal microscope.
Image Z-stacks were analyzed using Imaris 9.2.1 software. Optical density intensity sum values for each stack/channel were divided by corresponding data volumes and the resulting values were normalized to the number of specifically labeled cells within each stack. There were no significant differences in the number of cells between vehicle and KP-treated groups.
δ-Catenin DNA Constructs, Transgenesis Vectors
Plasmid containing human δ-catenin (CTNND2, NM_001288717) open reading frame was obtained from GeneCopeia (EX-A4285-M02) and cloned into pCMV-ENTER vector (Origene PS100001). Site-directed mutagenesis using Phusion DNA Polymerase enzyme (Thermofisher F549L) in conjunction with complementary primers bearing the specific mutation were used to generate the S276A S-catenin mutation S276A. PCR was followed by Dpn1 enzyme digestion to remove parental plasmid DNA. All constructs were verified by sequencing. pCS-CMV-tdTomato plasmid was obtained from Addgene (cat. #30530).
Plasmid pAAV-hSyn-eNpHR 3.0-EYFP from Addgene (26972) was cut with AgeI and HindIII enzymes to excise the eNpHR 3.0-EYFP open reading frames. The control tdTomato open reading frame as well as the wild-type and mutant delta catenin open reading frames were generated with AgeI and HindIII ends by PCR and subsequently inserted into the AgeI/HindIII digested pAAV-hSyn plasmid. Orientation and sequence fidelity in the final constructs were verified by PCR and sequencing. AAV9 particles were packaged by the Duke University Viral Vector Core facility and were used at a titer of 1012 viral genome copies per mL.
KCC2 Promoter Luciferase Reporter Assays
A fragment of the mouse KCC2 gene promoter (position −2052 kbp to +476 kbp) was amplified from genomic DNA prepared from cultured mouse primary glial cells. A 2.5 kb PCR fragment was cloned into the pGL4.17-Basic Vector (Promega) to generate the KCC2 promoter reporter construct. TCF and Kaiso binding sites were identified in this fragment. Using wild-type construct pGL4.17-KCC2 as a template, site-directed mutagenesis using Phusion DNA Polymerase enzyme (Thermo Fisher F549L) in conjunction with complementary primers bearing the specific mutation were used to mutate the Kaiso and TCF DNA-binding sites. PCR was followed by Dpn1 enzyme digestion to remove parental plasmid DNA. All constructs were verified by sequencing.
N2a cells were grown to 90% confluency in 24-well dishes in 0.4 mL of medium (DMEM, 2% Fetal Bovine Serum, 2 mM glutamine, 1% Non-essential amino acids, and 1% Penicillin/Streptomycin). Cells were transiently transfected using TurboFect reagent (Thermo Fisher R0531), with 500 ng of the pGL4.17-constructs plus 20 ng of the control Renilla plasmid (Promega, E2231) to normalize for transfection efficiency. Twenty-four hours after transfection, luminescence was measured using the Dual-Luciferase® Reporter Assay System (Promega) in a microplate luminometer (Veritas, Turner Biosystems). Mutant promoters were compared to WT, and the response of the respective promoter to KP (400 nM) was measured. Three independent transfection experiments were carried out and LUC assays were done in triplicates for each transfection. RLU is expressed as firefly luciferase activity relative to Renilla LUC activity.
Chemicals
kenpaullone compound was synthesized by the Duke Small Molecule Synthesis Facility to >98% purity, verified by LC/MS. CLP257, ICG-001, TWS119, CHIR99201, and VU0240551 were obtained from Tocris. GW801372X, GW778894X, GW300660X, GW779439X, and GW305178X were supplied by the Structural Genomics Consortium (SGC) at UNC-Chapel Hill.
Animals
C57bl/6J male mice (10-12 weeks old) were obtained from The Jackson Lab (Bar Harbor, Me.). KCC2-LUC mice were generated by the Liedtke Lab at Duke University and continued as a line within our mouse colony. All animal procedures were approved by The Duke University IACUC.
RT-qPCR
Total RNA was isolated from cultured cell samples using Directzol RNA miniprep kit (ZymoResearch). The protocol includes DNAse digestion to exclude genomic DNA from preparations. Total RNA (1 μg) was reverse transcribed using oligo primers (dT) and SuperScriptIII first-strand synthesis kit (Invitrogen). Gene expression was assessed by quantitative real-time PCR using 2×SYBR Green Master Mix (Qiagen) and a three-step cycling protocol (anneal at 60° C./elongate at 72° C., denature at 95° C.). Specificity of primers was verified by dissociation/melting curve for the amplicons when using SYBR Green as a detector. All reactions were performed in triplicates. The amount of target messenger RNA (mRNA) in the experimental group relative to that in the control was determined from the resulting fluorescence and threshold values (Ct) using the ΔΔCt method. βIII-tubulin was used as housekeeping gene.
RT-qPCR from Cultured Neuronal Cells and Microdissected Spinal Cord Dorsal Horn
oligodT-initiated reverse transcription of 1 μg total RNA, DNA-se treated, was subjected to RT-qPCR using primers specific for KCC2 for rat and KCC2 for human sequences, normalized for neuronal βIII-tubulin, as previously described (Yeo et al., 2009, J Neurosci 29:14652-14662). For cultured neurons, total RNA was extracted from pelleted cells, and for spinal cord tissue it was extracted from microdissected lumbar spinal cord dorsal horn.
Behavioral Assessments
For pain-related behavior, mechanical allodynia was assessed with von Frey filaments (Ugo Basile, Italy). The mice were placed on a 5×5-mm wire-mesh grid floor in individual compartments to avoid visual stimulation and allowed to adapt for 0.5 h prior to the von Frey test. The von Frey filament was then applied to the middle of the plantar surface of the hind paw with 5 g force. The withdrawal responses following the hind paw stimulation were measured at least three times, and the mechanical allodynia, which was defined as an increase in the number of withdrawal responses to the stimulation, was compared.
In addition, paws were stimulated with heat from underneath applied by an infrared beam (Hargreaves' test apparatus, IR level 40; Ugo Basile), and withdrawal latencies were recorded.
To assess scratching behavior as a behavioral correlate of itch, mice were shaved at the dorsal neck where topical application of 0.5% DNFB was applied (day 1). At days 5, 7, 9, and 11, mice received intraperitoneal (i.p.) injection of either KP or vehicle followed by 0.25% DNFB topical applications 4 hr later. On day 12, mice were allowed to acclimate to a Plexiglas chamber for at least 30 mins before performing itch behavior test. Scratching behavior was recorded by a Panasonic video camera for a 30-min observation period. Hind limb scratching behavior directed toward the shaved area at the nape of neck was observed. One scratch is defined as a lifting of the hind limb toward the injection site and then a replacing of the limb back to the floor, regardless of how many scratching strokes take place between those two movements. Behavioral analysis was conducted by observers blinded to treatment procedure. DNFB-induced scratching behavior was recorded on day 12 for 1 h. One scratch bout is defined as a lifting of the hind limb toward the injection site and then a replacing of the limb back to the floor, regardless of how many scratching strokes take place between those two movements. To determine the effect of KP on scratching behavior induced by DNFB, 30 mg/kg of KP was i.p. injected 20 min before each of DNFB challenge from day 5 to day 11.
For assessment in rotarod (RR), all animals received training prior to experiments; mice were placed on the RR apparatus set in an accelerating rotational speed mode (3-30 rpm, 300 s max) per trial. Following training, the average time to fall from the rotating cylinder over three trials was recorded as baseline latency (4-40 rpm, 300 s max/trial). Mice were injected daily with either vehicle or drug compounds before RR tests. Latency to fall was measured (4-40 rpm, 300 s max/trial (inter-trial interval is at least 15 min). The average latency to fall from the rod was recorded for each animal.
Conditioned place preference (CPP) was conducted using a CPP box, which consists of two conditioning chambers distinguished by visual and sensory cues, along with a small buffering chamber. All mice received a 3-day preconditioning habituation period with free access to both conditioning chambers and the time spent in each chamber was recorded for 15 min on day 3 after habituation. On conditioning days (day 4-10), mice first received the vehicle control (i.p. 5% DMSO, 5% Tween-80 in normal saline) paired with a randomly chosen chamber in the morning. After 4 hours, mice received KP (i.p. 30 mg/kg) or vehicle, paired with the other chamber. During the conditioning, mice were allowed to stay only in the paired chamber for 15 min without access to other chambers. On test day (d11), mice were placed in the buffering chamber with free access to both conditioning chambers and choice behavior was recorded for 15 min. The CPP scores were calculated as post-conditioning time minus preconditioning time spent in the paired chamber.
Chromatin Immunoprecipitation
ChIP assay was carried out as described previously (Yeo et al., 2009, J Neurosci 29:14652-14662; Yeo et al., 2013, Proc Natl Acad Sci USA 110:4315-4320). Primary cortical neurons (0.7×106) were used for each ChIP experiment. Cells were crosslinked with 1% formaldehyde for 30 min, washed twice with cold PBS, resuspended in lysis buffer [1% SDS, 10 mm EDTA, and 50 mm Tris-HCl, pH 8.0, with protease inhibitor cocktail (Roche)], and sonicated for 15 s pulses. The lysates were clarified by centrifugation at 10,000 rpm for 10 min at 4° C. in a microcentrifuge. One-tenth of the total lysate was used as input control of genomic DNA. Supernatants were collected and diluted in buffer (1% Triton X-100, 2 mm EDTA, 150 mm NaCl, 20 mm Tris-HCl, pH 8.0, and protease inhibitor cocktail) followed by immunoclearing with 1 mg of salmon sperm DNA, 10 ml of rabbit IgG, and 20 ml of protein A/G-Sepharose (Santa Cruz Biotechnology) for 1 h at 4° C. Immunoprecipitation was performed overnight at 4° C. with 2 mg of each specific antibody. Precipitates were washed sequentially for 10 min each in TSE1 buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 150 mm NaCl, and 20 mm Tris-HCl, pH 8.0), TSE2 (TSE1 with 500 mm NaCl), and TSE3 (0.25 m LiCl, 1% NP-40, 1% deoxycholate, 1 mm EDTA, and 10 mm Tris-HCl, pH 8.0). Precipitates were then washed twice with 10 mm Tris/0.1 mm EDTA, pH 7.8 and extracted with 1% SDS containing 0.1 m NaHCO3. Eluates were pooled and heated at 65° C. for 4 h to reverse formaldehyde crosslinking. DNA fragments were purified with Qiagen Qiaquick spin kit. For ChIP PCR, 1 l of a 25 l DNA extraction was used.
Immuno-Cytochemistry of Cultured Neurons
Immunocytochemistry labeling of cultured neuronal cells was carried out as previously described (Liedtke et al., 2013, Small 9, 1066-1075; Yeo et al., 2009, J Neurosci 29:14652-14662; Yeo et al., 2013, Proc Natl Acad Sci USA 110:4315-4320). Primary antibodies are shown in the antibody table. Anti-KCC2 primary antibodies were validated with developing rat primary cortical neurons; we observed an increase in staining pattern that tightly matched increase of KCC2 mRNA expression (Liedtke et al., 2013, Small 9:1066-1075; Yeo et al., 2009, J Neurosci 29:14652-14662.; Yeo et al., 2013, Proc Natl Acad Sci USA 110:4315-4320). Secondary antibodies used were goat anti-mouse IgG Alexa Fluor 594 (Invitrogen A11032) and goat anti-rabbit IgG Alexa Fluor 594 (Invitrogen A11012). DAPI stain was obtained from Sigma Aldrich (D9542). Stained cells were observed using an inverted confocal microscope (Zeiss LSM780).
We obtained stacks of images recorded at 0.35 μm intervals through separate channels with a 63× oil-immersion lens (NA, 1.40, refraction index, 1.45). Zen software (Zeiss) was used to construct composite images from each optical series by combining the images recorded through the different channels, and the same software was used to obtain Z projection images (image resolution: 1024×1024 pixels; pixel size: 0.11 μm). ImageJ was used for morphometry.
Spinal Cord Immuno-Histochemistry
All mice were deeply anaesthetized with isoflurane and then transcardially perfused with ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (4% PFA). Dissected spinal cord samples were then post-fixed overnight in 4% PFA at 4° C., cryoprotected in a 20% sucrose solution in PBS at 4° C., frozen in Tissue-Tek OCT (Sakura), and stored at −80° C. until sectioning. Samples were sectioned at 20 μm using a cryostat (Microm HM 505N). The sections were blocked with 2% bovine serum albumin (BSA) in PBS with 0.3% Triton X-100 (Blocking solution) at room temperature for 1 h. The sections were treated with primary antibody in blocking solution at 4° C. overnight. The sections were washed three times followed by secondary antibody treatment at 4° C. for 2 hours. Anti-KCC2 antibody was validated as described above for immuno-cytochemistry. The goat anti-rabbit IgG Alexa Fluor 488 was obtained from Invitrogen (A-11008). Morphometry was conducted using ImageJ with region-of-interest Rexed laminae I-II.
Chloride Imaging
Chloride imaging of primary cultured cortical neurons was conducted as previously described (Kuner and Augustine, 2000, Neuron 27:447-459; Liedtke et al., 2013, Small 9:1066-1075; Yeo et al., 2009, J Neurosci 29:14652-14662). A Clomeleon expression plasmid was transfected into primary cortical neurons by electroporation (Amaxa Nucleofector Device). Transfected neurons were verified by yellow fluorescent protein (YFP) fluorescence, and ratiometric images (excitation at λ=434 nm, dual emission at λ=485 and 535 nm; for resting chloride, six stable frames at a rate 12 of per minute were captured, which were averaged) were acquired using RATIOTOOL program. Calibration of Clomeleon signals (535 nm/485 nm emission ratio) was performed by using tributyltin-nigericin to establish a standard curve, which was then normalized for measured intraneuronal pH to take into account the pH sensitivity of Clomeleon.
Spinal Cord Dorsal Horn Electrophysiology
For spinal cord slice preparation, adult (5-7 weeks) male mice were anesthetized with urethane (1.5-2.0 g/kg, i.p.). The lumbosacral spinal cord was microsurgically removed and submerged into ice-cold dissection media which was saturated with 95% O2 and 5% CO2 at room temperature. After extraction and still under anesthesia, animals were euthanized. Transverse slices (300-400 m) were cut using a vibrating microslicer (VT1200s Leica). The slices were incubated at 32° C. for at least 30 min in regular artificial cerebrospinal fluid (aCSF), equilibrated with 95% O2 and 5% CO2.
The following solutions were used: Dissection solution: Sucrose 240 mM, NaHCO3 25 mM, KCl 2.5 mM, NaH2PO4 1.25 mM, CaCl2 0.5 mM, MgCl2 3.5 mM (Cheng et al., 2017, Nature neuroscience 20, 804-814). Regular artificial cerebrospinal fluid (ACSF): NaCl 117 mM, KCl 3.6 mM, MgCl2 1.2 mM, CaCl2 2.5 mM, NaHCO3 25 mM, NaH2PO4 1.2 mM, glucose 11 mM. The pH value of ACSF or dissection solution was adjusted to 7.4 when saturated with the gas. Normal intrapipette solution (pH 7.2 and 310 mOsm): K-methylsulfate 115 mM, KCl 25 mM, MgCl2 2 mM, HEPES 10 mM, GTP-Na 0.4 mM and Mg-ATP 5 mM.
Electrophysiological recordings were conducted as follows. A slice was placed in the recording chamber and completely submerged and superfused at a rate of 2-4 ml/min with aCSF saturated with 95% O2 and 5% CO2 at room temperature. Perforated patch-clamp was used to avoid alteration of the [Cl—]i. To measure the chloride equilibrium potential (EC1), gramicidin D (80 μg/mL with 0.8% DMSO final concentration, from an 8 mg/mL stock in DMSO) was added to the intrapipette solution, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM), DL-2-amino-5-phosphonovaleric acid (APV, 50 μM), and tetrodotoxin (TTX, 0.5 μM) were added to the aCSF solution. The tip of the patch pipette was filled with the normal intrapipette solution while the rest of the pipette contained the gramicidin-containing solution. After forming a seal on the membrane, we waited ˜30 min for the gramicidin to induce sufficient cation-selective pores in the membrane and lowered the series resistance to below 100 MΩ. Membrane potential measurements were corrected for liquid junction potential, which was measured as in (Jiang et al., 2014, Journal of neurophysiology 111:991-1007). GABA (1 mM) was puffed locally and instantaneously, and the puff pipette was aimed toward the recording pipette. To determine the reversal potential of GABA-evoked currents, voltage ramps were applied from +8 to −92 mV over 200 ms at a holding potential of −42 mV. Since the voltage ramp might elicit a basal current, a control voltage ramp was applied, and 1 min later GABA was puffed followed by another voltage ramp (Billups and Attwell, 2002, The Journal of physiology 545:183-198). The reversal potential was analyzed as previously described (Billups and Attwell, 2002, The Journal of physiology 545:183-198).
Signals were acquired using an Axopatch 700B amplifier and analyzed with pCLAMP 10.3 software. Only neurons with resting membrane potential <−50 mV and stable access resistance were included.
Drug Affinity Responsive Target Stability (DARTS) assay
Cultured primary rat cortical neurons were treated with either vehicle DMSO (0.1%) or 20 μM KP for 30 h. Cells were lysed in ice-cold lysis buffer (Tris.Cl pH8 50 mM, NaCl 150 mM, NP40 0.5%, N-dodecyl-b-D-maltoside 0.5%, Phosphatase Inhibitor (Pierce #88667) and Protease Inhibitor (Roche #11836153001)). Protein concentrations were determined by Bio-Rad DC Protein Assay kit using bovine albumin as standard. All steps were performed on ice. Samples were warmed to room temperature and digested with pronase (final concentration 1:500) for 30 min at 30° C. Digestion was halted using 0.5M EDTA. Only proteins not bound to KP were digested. The protein mixture was dialyzed using dialysis cassettes (Thermo Fisher 66203, 2K MWCO) and analyzed by LC-MS/MS method to identify proteins that are bound to KP, the latter step carried out in the Duke Proteomics Core Laboratory.
Kinome Analysis
Cultured rat primary cortical neurons were treated with either vehicle DMSO (0.1%) or 1 μM KP for 1 h/24 h. Cells were lysed in non-detergent-containing buffer (Tris.Cl pH8 50 mM, NaCl 150 mM, 0.5% Phosphatase Inhibitor (Pierce #88667) and Protease Inhibitor (Roche #11836153001)). 500 μL was removed and solid urea was added to a final concentration of 8M. Samples were sonicated for further solubilization. After clearing of insoluble material by centrifugation, protein concentration was measured by Bradford assay. 250 μg of total protein was removed from each sample and solubilization buffer was added to normalize all samples to 0.93 μg/μL protein. Samples were then spiked with bovine alpha-casein to 30 fmol/μg of total protein. Samples were reduced with 10 mM DTT at 32° C. for 45 min and then alkylated with 20 mM iodoacetamide at room temperature for 30 min. Samples were trypsin digested at 1:25 (enzyme-to-protein) overnight at 32° C. Following acidification with TFA to pH 2.5, samples were subjected to a C18 solid-phase extraction cleanup. Eluted peptides were split 80% for phosphopeptide analysis and 20% reserved for unbiased differential expression. The phosphopeptide fraction (200 μg) was then frozen and lyophilized prior to phosphopeptide enrichment.
TiO2 Enrichment. Samples were resuspended in 65 μL of 1M glycolic acid in 80% MeCN/1% TFA and were enriched on TiO2 resin using a 10 μL GL Sciences microliter TiO2 spin tips following an established protocol (www.genome.duke.edu/cores/proteomics/samplepreparation/documents/GL_SpinColumnPr otocol_bmr_ejs_mt_061713.pdf). After elution and acidification, samples were lyophilized to dryness and resuspended in 100 μL of 0.15% TFA in water. After cleanup using a C18 STAGE tip, and resuspension in 2% acetonitrile, 0.1% TFA, 10 mM citric acid samples were quantified.
Quantitative analysis of Phosphopeptide Enriched Samples. Quantitative LC-MS/MS was performed in singlicate (4 uL=33% of the total sample each injection) for phosphopeptide-enriched samples using a nanoAcquity UPLC system (Waters Corp) coupled to a Thermo QExactive Plus high resolution accurate mass tandem mass spectrometer (Thermo) via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C18 300 mm Ř180 mm trapping column for 6 min at 51/min (99.9/0.1 v/v water/acetonitrile 0.1% formic acid), after which the analytical separation was performed on a 1.7 μm Acquity BEH130 C18 75 mm Ř250 mm column (Waters Corp). Peptides were held at 3% acetonitrile with 0.1% formic acid for 5 min and then subjected to a linear gradient from 3 to 30% acetonitrile with 0.1% formic acid over 90 min at a flow rate of 400 nL/min at 55° C. Data collection on the QExactivePlus mass-spec was performed in a data-dependent acquisition (DDA) mode following protocol of the manufacturer.
Statistics
All data are expressed as mean±SEM. Differences between groups were evaluated using two-tailed Student's t test (experimental against sham control), or in the case of multiple groups, one-way ANOVA followed by post-hoc Bonferroni test. The criterion for statistical significance is p<0.05.
To identify KCC2 gene expression enhancing compounds, cultured primary cortical neurons were cultured from KCC2-luciferase(LUC)-knockin (KCC2-LUCki) mice (Liedtke et al., 2013, Small 9:1066-1075; Yeo et al., 2013, Proc Natl Acad Sci USA 110:4315-4320) and used LUC metrics as readout for activity of the proximal KCC2 promoter (2.5 kB (Yeo et al., 2009, J Neurosci 29:14652-14662)), which drives LUC in this mouse transgenic line. A Z′ factor of 0.94 was obtained. This strategy will not select for long-range enhancers of KCC2 gene expression that act outside the 2.5 kB core KCC2 promoter. 1057 compounds, contained in two NCI libraries (
Of these, kenpaullone (KP) (9-bromo-7,12-dihydro-indolo [3,2-d] [1]benzazepin-6(5H)-one), a GSK3/CDK kinase inhibitor, was identified as one of the promising compound for further study based on its previous record of neuroprotection in translationally relevant preclinical models (Liu et al., 2016, Cell reports 14:115-128; Reinhardt et al., 2019, Stem Cell Reports 12:502-517; Skardelly et al., 2011, Neuroscience 29:543-547; Yang et al., 2013, Cell Stem Cell 12:713-726).
These data establish that (i) KP enhanced KCC2 gene expression in rat and mouse primary cortical neurons (
In view of this discovery, in vivo analgesic effects of KP were addressed next.
It was found that KP showed analgesic effects in two preclinical mouse models of pathologic pain: nerve injury-induced neuropathic pain, implemented by peripheral nerve constriction, and inflammatory pain, induced by peripheral tissue injection of complete Freund's adjuvans (CFA) (
Next, whether the analgesic effects of KP were mediated centrally at the spinal cord level was assessed. KP was injected intrathecally (i.t; 30 μg) and reduced mechanical allodynia in mice with nerve constriction injury was observed (
Based on these findings, KP's anti-pruritic effects were assessed because inhibitory transmission in the SCDH plays an important role in chronic pruritus (Akiyama et al., 2015, Pain 156:1240-1246.; Bourane et al., 2015, Science 350:550-554; Braz et al., 2017, Prog Brain Res 231:87-105; Braz et al., 2014, J Clin Invest 124:3612-3616; Koch et al., 2018, Annual review of physiology 80:189-217; Mishra and Hoon, 2015, Handbook of experimental pharmacology 226:151-162). In a 2,4-dinitrofluorobenzene (DNFB) chronic contact dermatitis model (Zhang et al., 2015, Cell Physiol Biochem 35:1023-1033), daily i.p injections of KP (30 mg/kg) significantly reduced robust scratching behavior of sensitized sites (
With these beneficial effects of KP on pathologic pain and itch, whether KP had undesirable effects on the CNS in terms of sedation, impairment of motor stamina, balance and coordination was assessed. Rotarod testing (Heyser et al., 2013, Physiol Behav 118:208-211) of KP-treated mice (10, 30 mg/kg; i.p.) revealed that KP does not induce unwanted side effects (
With the aforementioned findings and in view of the SCDH as the likely site of analgesic action of KP (Coull et al., 2003, Nature 424:938-942; Gagnon et al., 2013, Nat Med 19:1524-1528), SCDH KCC2 expression and function in nerve injury and response to KP was assessed. It was found that in the SCDH, KP repaired attenuated KCC2 expression caused by nerve injury, at both the mRNA and protein levels (
Next, whether repair of attenuated KCC2 expression was associated with re-normalization of chloride reversal potential after application of GABA (EGABA) was assessed. Prior to interrogation of spinal cord slice preparations at 72 h post-injury, robust mechanical allodynia of young mice by peripheral nerve constriction injury and its almost complete behavioral reversal by systemic treatment with KP at 30 mg/kg was shown (
These findings set up a compelling rationale to deconstruct the cellular mechanism of action of KP that accounts for its effects in SCDH neurons and thus its sensory effects. These studies were conducted in primary cortical neurons because 1) these neurons were used for the initial screen, 2) there was rodent-human similarity in terms of the effects of KP on KCC2 KCC2 gene expression, and 3) the effects of KP on KCC2 gene expression and KCC2 chloride transporter function in these neurons were highly similar to our findings in SCDH lamina-II neurons, which cannot be cultured as readily for mechanistic cellular studies.
First, the question of whether KP inhibiting GSK3ß or CDKs is responsible for increasing expression of KCC2 was addressed. Using a set of GSK3 inhibitors different from KP, increased expression of KCC2 was shown. In contrast, a suite of CDK-inhibitory compounds did not increase (but decreased) expression of KCC2 (
A drug affinity responsive target stability (DARTS) assay (Lomenick, et al., 2009, Proc Natl Acad Sci USA 106:21984-21989) was used to study direct binding of KP to GSK3ß (
To investigate the relevance of δ-cat residue S276, N2a neural cells were studied because these cells transfect at higher efficiency than primary cortical neurons. In our cultures, N2a cells expressed neuronal ßIII-tubulin in elongated processes as determined by immune-labeling as an indicator of their neuronal differentiation. Furthermore, nuclear transfer of δ-cat was significantly enhanced upon KP treatment in N2a cells transfected with human δ-cat(WT) (micrograph images not shown). This increase in nuclear transfer was very similar to the trafficking we recorded in primary cortical neurons, thus validating the cell line. To determine the function of δ-cat residue S276 we then transfected δ-cat(S276A), which resulted in significantly increased nuclear transfer of δ-cat(S276A), using morphometry. Of note, there was no increase of nuclear transfer of δ-cat(S276A) upon KP treatment (
Thus, δ-cat(S259/S276) (rat/human) is very likely a relevant phosphorylation site in δ-cat and a GSK3ß kinase target in neurons. Inhibition of GSK3ß or rendering S276 phosphorylation-resistant enhances nuclear transfer of δ-cat, also of its binding partner ß-cat. Furthermore, a selective catenin-inhibitor attenuated KCC2 gene expression dose-dependently (
Consequently, catenins effects on the KCC2 promoter was explored. Two Kaiso binding sites (potential sites for delta-cat (Rodova, et al. 2004, Mol Cell Biol 24:7188-7196; Dai, et al. 2011, Cancer science 102:95-103) were identified in the KCC2 proximal promoter using computational methods (Aerts, et al., 2005, Nucleic Acids Res 33:W393-396). Two Kaiso binding sites bracketed the transcriptional start site (TSS) of the KCC2 gene (
Promoter expression constructs were built with rationally-targeted deletions to interrogate the effects of the Kaiso- and TCF-binding sites on activity of the KCC2 promoter and to determine if this activity was regulated by KP. For ease of transfection, key to this method, N2a neural cells were again used. The 5′ and 3′ delta-cat Kaiso binding sites functioned in repressive and enhancing manners, respectively (
These data suggest that δ-cat, a kinase target of GSK3ß in CNS neurons, traffics to the nucleus increasingly upon GSK3ß inhibition. In the nucleus, δ-cat interacts with the KCC2 promoter to enhance KCC2 expression via two Kaiso DNA-binding sites. ⊕-cat co-traffics to the nucleus with δ-cat in response to KP. Beta-cat is not a significant neuronal GSK3ß kinase target, and plays an ancillary role in enhancement of KCC2 gene expression.
Next, the question of whether δ-cat, when expressed as a spinal transgene in sensory relay neurons, will facilitate analgesia in nerve constriction injury was assessed. A δ-cat transgene increases KCC2 expression in N2a neural cells was observed, and that KCC2 expression levels were slightly elevated when using δ-cat(S276A) (
AAV9 vectors harboring human δ-cat and δ-cat(S276A), driven by the minimal human neuronal synapsin promoter (huSyn (Liu, et al. 2008, BMC Biotechnol 8:49) were constructed. AAV9 and huSyn were used because in a previous in-depth study, AAV9 harboring fluorescent reporter driven by huSyn, upon i.t injection, readily transduced spinal neurons and spared DRG primary afferent neurons (Haenraets, et al., 2017, Journal of neurochemistry 142:721-733). Synapsin-tdTomato was used as control and injected 5×109 viral genomes (5 μL; i.t) of each construct. Assessment of tdTomato fluorescence 3d post injection revealed spinal transgenesis that was evenly manifesting in the SCDH (
Discussion
Evidence that reduced expression of the neuronal chloride extruding transporter, KCC2, contributes to a diversity of CNS diseases led us to seek a small molecule which increased its expression. Using measures of KCC2 promoter activity, KCC2 mRNA abundance, and [Cl—]i, an unbiased screen of two NCI libraries containing 1057 compounds that inhibit growth of transformed cells was conducted. This screen identified kenpaullone (KP), a GSK3/CDK kinase inhibitor with neuroprotective properties (Liu et al., 2016, Cell reports 14:115-128; Reinhardt et al., 2019, Stem Cell Reports 12:502-517; Schultz et al., 1999, J Med Chem 42:2909-2919, Skardelly et al., 2011, Neuroscience 29:543-547; Yang et al., 2013, Cell Stem Cell 12:713-726; Zaharevitz et al., 1999, Cancer research 59:2566-2569). These studies of KP revealed at least the following principal findings: 1) KP enhances KCC2 KCC2 gene expression in a concentration-dependent manner and lowers [Cl—]i in cultured mouse, rat, and human neurons; 2) Systemic administration of KP to mice attenuates measures of nerve injury pain and chronic itch in preclinical models; 3) Intrathecal administration of KP to mice attenuates nerve injury pain depending on spinal KCC2 chloride transporter activity; 4) Systemic administration of KP to mice with nerve injury enhances KCC2 gene expression in SCDH neurons and shifts the GABA-induced chloride reversal potential to more negative and electrically stable measures; and 5) The mechanism by which KP enhances KCC2 gene expression is by binding to and inhibiting GSK3ß, inhibiting phosphorylation of δ2-cat at position S259 in rat (S276 in human), which increases nuclear transfer of δ2-cat. In the nucleus, δ2-cat binds to and enhances the KCC2 promoter via two Kaiso-binding regulatory sites. Transferring this mechanism from neural cells to the live animal, spinal transgenesis of δ2-cat(S2276A) attenuates nerve injury pain in mice. Thus, KP and the new GSK3ßδ2-cat
Kaiso
KCC2 signaling pathway may represent a strategic bridge-head for therapeutics development for treatment of pathologic pain. Beyond pain, this could also apply to other neurologic and mental health conditions in which restoration of KCC2 function is important, such as epilepsy, traumatic spinal cord and brain injury, neurodegeneration and neurodevelopmental disorders. This proposed analgesic mechanism is summarized in
Compounds from two NCI libraries that can interfere with gene regulation in CNS neurons were identified. A substantial number of the molecules contained in these libraries can regulate growth of rapidly dividing cells by interfering with chromatin and via additional epigenetic mechanisms. Therefore, these compounds can act similarly in non-dividing CNS neurons such as primary developing cortical neurons. These neurons helped identify compounds that can activate the KCC2 promoter, and KP was selected as the “winner” compound for in-depth exploration. Though KP is predicted to have multiple targets in CNS neurons, the data shown herein provide evidence that KP binds to neuronal GSK3ß and not to CDKs. In addition, it is known that KP inhibits GSK3ß with highest potency from amongst known targets (Knockaert et al., 2002, The Journal of biological chemistry 277:25493-25501; Kunick et al., 2004, Bioorg Med Chem Lett 14:413-416; Schultz et al., 1999, J Med Chem 42:2909-2919). The results demonstrated that GSK3ß inhibition by KP directly upregulates KCC2 gene expression via the δ-catenin-Kaiso pathway. This mechanism of a GSK3-inhibitory compound has not been reported previously. Of note, this novel concept held true in primary human neurons in which KP also enhanced synaptic maturation. Although KP can inhibit other kinases, the data suggest that inhibition of GSK3ß and subsequent enhancement of KCC2 gene expression via S-catenin are very important, perhaps dominant mechanisms of action of KP as it attenuates pathologic pain. Additionally, S-cat-Kaiso likely affects multiple neuronal genes, but the data suggest that enhanced KCC2 gene expression and KCC2 function are the major analgesic effector mechanisms of KP. Another argument, mechanistically weaker but translationally relevant, is the absence of unwanted effects of our KCC2 expression-enhancing strategy on choice behavior, motor stamina, and coordination. Effective targeting of multiple pathways would likely impact these behaviors. However, the behavioral profile for KP was similarly benign as other KCC2 expression-enhancing compounds (Gagnon et al., 2013, Nat Med 19:1524-1528).
The screening strategy addressed a fundamental problem of pathological pain. This vastly unmet medical need, rooted in its chronicity, is driven by genetic reprogramming, which results in a maladaptive phenotype (Bai et al., 2015, Translational research:the journal of laboratory and clinical medicine 165:177-199; Doyon et al., 2013, Expert review of neurotherapeutics 13:469-471; Kuner, 2010, Nat Med 16:1258-1266; Liang et al., 2015, Epigenomics 7:235-245; Sommer, 2016, Science 354:588-592). A key event is attenuated expression of KCC2 because of its relevance for inhibitory transmission in pain-relevant neural circuits (Coull et al., 2003, Nature 424:938-942; Gagnon et al., 2013, Nat Med 19:1524-1528; Kahle et al., 2014, JAMA neurology 71:640-645; Li et al., 2016, Cell reports 15:1376-1383; Mapplebeck et al., 2019, Cell reports 28:590-596 e594; Price et al., 2005, Curr Top Med Chem 5:547-555). This has also been postulated in other pathologic conditions of the CNS as a general pathogenic feature (Hwang and Zukin, 2018, Current opinion in neurobiology 48:193-200; Lardenoije et al., 2015, Progress in neurobiology 131:21-64). Regulation of KCC2 gene expression by GSK3ß and its kinase target δ-cat is a novel insight of the study described herein. This concept will permit rational exploration of links between GSK3ß
δ-cat and attenuated KCC2 KCC2 gene expression and the resulting malfunction of inhibitory neurotransmission in several other relevant neurologic and psychiatric conditions such as Alzheimer's Disease and other neurodegenerative diseases, psychoses, traumatic brain/spinal cord injury, Rett Syndrome, Autism Spectrum Disorders, and epilepsy (Boulenguez et al., 2010, Nat Med 16:302-307; Chen et al., 2018, Cell 174:521-535 e513; Ferando et al., 2016, Nature neuroscience 19:1197-1200; Freund and Meskenaite, 1992, Proc Natl Acad Sci USA 89:738-742; Huberfeld et al., 2007, The Journal of neuroscience:the official journal of the Society for Neuroscience 27:9866-9873; Hyde et al., 2011, J Neurosci 31:11088-11095; Kahle et al., 2014, EMBO Rep 15, 766-774; Tang et al., 2016, Proc Natl Acad Sci USA 113:751-756; Tao et al., 2012, J Neurosci 32:5216-5222). KP was selected because of its previously reported neuroprotective properties for spinal motoneurons, brainstem auditory relay neurons, and hypoxia-injured hippocampal neurons (Liu et al., 2016, Cell reports 14:115-128; Reinhardt et al., 2019, Stem Cell Reports 12:502-517; Skardelly et al., 2011, Neuroscience 29:543-547; Teitz et al., 2018, The Journal of experimental medicine 215:1187-1203; Winkelmann et al., 2015, Cell death & disease 6:e1776; Yang et al., 2013, Cell Stem Cell 12:713-726). There was a unifying mechanism of KCC2 expression enhancement by KP in these previous studies. Neuroprotective properties for a novel analgesic are welcome because chronic pain is associated with non-resolving neural injury mediated by neuroinflammation (Ji et al., 2018, Anesthesiology 129:343-366). Repurposing a GSK3ß-inhibitory compound as an analgesic reprogramming compound that upregulates KCC2 expression links chronic pathologic pain to neurodegeneration at both the basic science and translational neuroscience levels.
The work described herein characterized a novel strategy to treat chronic pathologic pain and also increases our basic understanding of pain and sensory transduction. With a focus on the enhanced expression of KCC2 in neurons as an analgesic strategy, a compound was utilized as a genomic reprogramming agent that reverts the expression of a key dysregulated gene. Of practical/translational importance, it was demonstrated that KP is non-sedative and does not affect motor stamina, coordination, or choice behavior. Furthermore, validated KP was targeted in human primary neurons. In aggregate, a repurposed compound, KP, was identified and a genetically-encoded cellular signaling pathway, GSK3ß
δ-cat
Kaiso
KCC2, which were not previously known to up-regulate KCC2. Both of these KCC2-enhancing approaches can be developed translationally into clinical neuroscience applications.
The finding that KP acts as an anti-pruritic in a chronic contact dermatitis model is novel and suggests that defective expression of KCC2 in the SCDH plays an important role in chronic inflammatory itch. This finding requires additional research to further elucidate the underlying cellular and neural circuit mechanisms. Validation in additional itch models will also facilitate translation to the clinic.
The approach as described herein for targeting of pain and itch is innovative. Enhanced KCC2 gene expression, based on KP treatment or δ-cat transgenesis as presented here, will complement direct enhancement of KCC2 chloride extrusion in targeting pathologic pain. Complementary use will help overcome recalcitrant lack of expression and function of KCC2 in pain relay neurons, as might be expected in clinical cases of “refractory” chronic pain. Clinical combination use of KCC2 expression enhancers with analgesic compounds that have different mechanisms of action will be advantageous as renormalized inhibitory transmission will cause improved effectiveness of other compounds, such as gabapentinoids.
One skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present disclosure described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the present disclosure. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the present disclosure as defined by the scope of the claims.
No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents form part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise.
The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references.
Additional Sequences:
Homo sapiens catenin delta 2 (CTNND2), transcript variant/isoform 1
Homo sapiens catenin delta 2 (CTNND2), transcript variant 2 (Acc.
Homo sapiens catenin delta 2 (CTNND2), transcript variant 3 (Acc.
Homo sapiens catenin delta 2 (CTNND2), transcript variant 4 (Acc.
Homo sapiens catenin delta 2 (CTNND2), transcript variant 6 (Acc.
Homo sapiens delta-catenin (Acc. No.: U96136.1); Transcript
This application claims priority to U.S. Provisional Patent Application Ser. No. 62/890,710, filed Aug. 23, 2019, the contents of which is hereby incorporated by reference in its entirety.
This invention was made with government support under Grant Nos. NS066307, DE11794, and DE022793, all from the National Institutes of Health (NIH). The Federal Government has certain rights to this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/047655 | 8/24/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62890710 | Aug 2019 | US |
