# Patent

## Title
Treating neurological disorders

## Abstract

Methods of treating neuronal disorders, such as mechanical neuronal traumas and neurodegenerative disorders, with TWEAK or a TWEAK receptor blocking agents are presented.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a division of U.S. application Ser.No. 13/222,097, filed Aug. 31, 2011 (abandoned), which is a Continuation of U.S. application Ser. No. 11/816,502, filed Oct. 3, 2008 (abandoned), which is a National Phase entry of PCT/US2006/005597 filed Feb. 17, 2006, which claims the benefit of U.S. Provisional Application No. 60/653,813filed Feb. 17, 2005, all of which are incorporated herein in their entirety by reference.
SEQUENCE LISTING
The instant application contains a sequence listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety.


BACKGROUND
Neurological disorders affect an estimated 50 million Americans each year, exacting an incalculable personal toll and an annual economic cost of hundreds of billions of dollars in medical expenses and lost productivity.
SUMMARY
TWEAK (TNT-like weak inducer of apoptosis) is a trimeric protein that is a TNF (Tumor Necrosis Factor) superfamily member. TWEAK mediates cellular responses by activating a cell surface receptor (referred to as a TWEAK receptor or TWEAK-R herein), such as the Fn14 protein. As further described below, blocking TWEAK/TWEAK-R interaction or activity is an effective and useful therapy for neuronal injury or pathology, e.g., in the CNS or peripheral nervous system. The therapy can be used to treat neuronal disorders such as mechanical neuronal traumas and neurodegenerative disorders. Examples of mechanical neuronal traumas include spinal cord injury (SCI) and traumatic brain injury (TBI). Examples of neurodegenerative disorders include amyotrophic lateral sclerosis (ALS), progressive bulbar palsy (PBP), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), Parkinson's Disease, Huntington's Disease (HD), and Alzheimer's Disease.
A variety of TWEAK and/or TWEAK receptor blocking agents can be administered to a subject to block a TWEAK/TWEAK-R interaction or activity. A “TWEAK/TWEAK-R blocking agent” or “agent that blocks a TWEAK/TWEAK-R interaction or activity” refers to an agent (e.g., any compound, e.g., an antibody or a soluble form of the TWEAK receptor) that at least partially inhibits an interaction or activity of a TWEAK or TWEAK receptor. For example, the agent at least partially inhibits an activity, e.g., binding of TWEAK to a TWEAK receptor, or the agent at least partially inhibits a nucleic acid encoding TWEAK or TWEAK receptor, e.g., to reduce TWEAK or TWEAK receptor protein expression.
In one embodiment, the agent reduces the ability of TWEAK to bind to Fn14 (a TWEAK receptor). The agent can be an antibody that binds to TWEAK or to Fn14, The antibody can be a full length IgG. In one embodiment, the antibody is human, humanized, or effectively human.
In one embodiment, the agent is a soluble loin of a TWEAK receptor, e.g., a human TWEAK receptor such as Fn14. The soluble form of the TWEAK receptor can be fused to an antibody Fc region (e.g., a human Fc region). For example, the soluble form of the TWEAK receptor includes a sequence at least 95% identical to amino acids 28-X1 of SEQ ID NO:2, where amino acid X1 is selected from the group of residues 68 to 80 of SEQ ID NO:2.
In one embodiment, a primary characteristic of the neuronal disorder is not demyelination or the presence of autoantibodies against myelin.
In one embodiment, the neuronal disorder is primarily characterized by in destruction or death of nerve cells, e.g., of motor neurons (e.g., ALS), of striatal neurons of basal ganglia and/or cortical neurons (e.g., Huntington's disease), of substantia nigra neurons (e.g., Parkinson's disease).
In one aspect, the disclosure features a method of treating spinal cord injury (SCI). The method includes administering a TWEAK/TWEAK-R blocking agent to a subject who has experienced or is at risk of experiencing a spinal cord injury, e.g., within the previous 48, 24, 12, or 6 hours. The agent can be administered in an amount and/or for a time to ameliorate at least one symptom of SCI, to reduce neuronal damage associated with SCI, and/or to reduce inflammation associated with SCI. The agent can be administered in combination with another treatment for a spinal cord injury, e.g., administering corticosteroids such as methylprednisolone or stabilization of the vertebrae of the spine. Further, the method can include evaluating the subject using a spinal cord injury assessment criterion, such as the American Spinal Injury Association assessment criteria and/or by performing a neurological exam. The method can include a step of identifying a subject who has sustained or is at risk of sustaining a spinal cord injury, e.g., detecting a spinal cord injury in a subject. A TWEAK/TWEAK-R blocking agent can be packaged in a container that has a label with instructions for use of the agent in treating a spinal cord injury. The disclosure also features an agent that blocks a TWEAK/TWEAK-R interaction or activity for use in treating a spinal cord injury and for the manufacture of a medicament for treating a spinal cord injury.
In one aspect, the disclosure features a method of treating traumatic brain injury (TBI). The method includes administering a TWEAK/TWEAK-R blocking agent to a subject who has sustained or experienced, or is at risk of experiencing or sustaining, a traumatic brain injury, e.g., within the previous 48, 24, 12, or 6 hours. The agent can be administered in an amount and/or for a time to ameliorate at least one symptom of TBI, to reduce neuronal damage associated with TBI, and/or to reduce inflammation associated with TBI. The agent can be administered in combination with another treatment for a traumatic brain injury, e.g., surgery to control bleeding in and around the brain, monitoring and controlling intracranial pressure, insuring adequate blood flow to the brain, or treating the body for other injuries and infection. Further, the method can include evaluating the subject using a traumatic brain injury assessment criterion, such as the Glasgow Coma Scale. The method can include a step of identifying a subject who has sustained or is at risk of sustaining a traumatic brain injury, e.g., by detecting a recent traumatic brain injury in a subject. A TWEAK/TWEAK-R blocking agent can be packaged in a container that has a label with instructions for use of the agent in treating a traumatic brain injury. The disclosure also features an agent that blocks a TWEAK/TWEAK-R interaction or activity for use in treating a traumatic brain injury or for the manufacture of a medicament for treating a traumatic brain injury.
In addition to therapeutic use in treating SCI and TBI, a TWEAK/TWEAK-R blocking agent can be used to treat other trauma to the central or peripheral nervous system, particularly mechanical trauma to the central nervous system. The treatment may also include a prophylactic component. For example, the blocking agent may be administered (locally or systemically) prior to, during, or after surgery, e.g., surgery that requires intervention in or near the central nervous system. In the case of unexpected traumas, it is frequently desirable to provide the blocking agent as soon as possible after the trauma event, e.g., within the previous 48, 24, 12, or 6 hours since the event. The blocking agent can also be used when grafting or implanting neuronal tissue (e.g., in a xeno-transplant, etc).
In one aspect, the disclosure features a method of treating amyotrophic lateral sclerosis (ALS). The method includes administering a TWEAK/TWEAK-R blocking agent to a subject who has or has been diagnosed as having symptoms of ALS or a predisposition to developing ALS. The agent can be administered in an amount and/or for a time to ameliorate at least one symptom of ALS, to reduce neuronal damage associated with ALS, and/or to reduce inflammation associated with ALS. The agent can be administered in combination with another treatment for ALS, e.g., riluzole. Further, the method can include evaluating the subject using an ALS assessment criterion, such as the Appel ALS Rating Scale. The method can include a step of identifying a subject who has ALS, e.g., detecting ALS in a subject. A TWEAK/TWEAK-R blocking agent can be packaged in a container that has a label with instructions for use of the agent in treating ALS. The disclosure also features an agent that blocks a TWEAK/TWEAK-R interaction or activity for use in treating ALS and for the manufacture of a medicament for treating ALS.
In one aspect, the disclosure features a method of treating Parkinson's Disease. The method includes administering a TWEAK/TWEAK-R blocking agent to a subject who has or has been diagnosed as having symptoms of Parkinson's Disease or a predisposition to developing Parkinson's Disease. The agent can be administered in an amount and/or for a time to ameliorate at least one symptom of Parkinson's Disease, to reduce neuronal damage associated with Parkinson's Disease, and/or to reduce inflammation associated with Parkinson's Disease. The agent can be administered in combination with another treatment for Parkinson's Disease, e.g., levodopa and the dopamine agonists ropinirole, pramipexole, and pergolide, MAO-B inhibitors (e.g., R(+)—N-propargyl-1-aminoindan). Further, the method can include evaluating the subject using a Parkinson's Disease assessment criterion, such as the Hoehn and Yahr Staging of Parkinson's Disease; the Schwab and England Activities of Daily Living assessment; or the Unified Parkinson's Disease Rating Scale. The method can include a step of identifying a subject who has Parkinson's Disease, e.g., detecting Parkinson's Disease in a subject, A TWEAK/TWEAK-R blocking agent can be packaged in a container that has a label with instructions for use of the agent in treating Parkinson's Disease. The disclosure also features an agent that blocks a TWEAK/TWEAK-R interaction or activity for use in treating Parkinson's Disease and for the manufacture of a medicament for treating Parkinson's Disease.
In one aspect, the disclosure features a method of treating Huntington's Disease. The method includes administering a TWEAK/TWEAK-R blocking agent to a subject who has or has been diagnosed as having symptoms of Huntington's Disease or a predisposition to developing Huntington's Disease. The agent can be administered in an amount and/or for a time to ameliorate at least one symptom of Huntington's Disease, to reduce neuronal damage associated with Huntington's Disease, and/or to reduce inflammation associated with Huntington's Disease. The agent can be administered in combination with another treatment for Huntington's Disease, e.g., bromocriptine, serotonin antagonists (e.g., tetrabenazin), tiapride (e.g., TIAPRIDAL®), or a NMDA receptor blocker. Further, the method can include evaluating the subject using a Huntington's Disease function assessment criterion, such as the Unified Huntington's Disease Rating Scale (UNDRS). The method can include a step of identifying a subject who has Huntington's Disease, e.g., detecting Huntington's Disease in a subject. A TWEAK/TWEAK-R blocking agent can be packaged in a container that has a label with instructions for use of the agent in treating Huntington's Disease. The disclosure also features an agent that blocks a TWEAK/TWEAK-R interaction or activity for use in treating Huntington's Disease and for the manufacture of a medicament for treating Huntington's Disease.
In one aspect, the disclosure features a method of treating Alzheimer's Disease. The method includes administering a TWEAK/TWEAK-R blocking agent to a subject who has or has been diagnosed as having symptoms of Alzheimer's Disease or a predisposition to developing Alzheimer's Disease. The agent can be administered in an amount and/or for a time to ameliorate at least one symptom of Alzheimer's Disease, to reduce neuronal damage associated with Alzheimer's Disease, and/or to reduce inflammation associated with Alzheimer's Disease. The agent can be administered in combination with another treatment for Alzheimer's Disease, e.g., tacrine (COGNEX®), donepezil (ARICEPT®), rivastigmine (EXELON®), galantamine (REMINYL®), memantine (NAMENDATM), nonsteroidal anti-inflammatory drugs (NSAIDS), statins, folic acid, gingko biloba, or vitamin E, B6, or B12. Further, the method can include evaluating the subject using an Alzheimer's Disease function assessment criterion, such as the Progressive Deterioration Scale (PDS) or the Mini-Mental State Examination (MMSE). The method can include a step of identifying a subject who has Alzheimer's Disease, e.g., detecting Alzheimer's Disease in a subject. A TWEAK/TWEAK-R blocking agent can be packaged in a container that has a label with instructions for use of the agent in treating Alzheimer's Disease. The disclosure also features an agent that blocks a TWEAK/TWEAK-R interaction or activity for use in treating Alzheimer's Disease and for the manufacture of a medicament for treating Alzheimer's Disease.
In addition to ALS, Huntington's Disease, Alzheimer's Disease, and Parkinson's Disease, a TWEAK/TWEAK-R blocking agent can be used to treat or prevent other neurological disorders including neurodegenerative disorders. These disorders include ones that cause damage to motor neurons, inter-neurons, and/or sensory neurons, or neurons of particular classes, e.g., dopamine producing neurons. Examples of such disorders include disorders that involve gradual degeneration of neuronal function, e.g., in the central or peripheral nervous system. Such degeneration can result from a variety of pathologies, including the accumulation of toxic structures such as neurofibrillary tangles, protein aggregates, prions, and plaques. Many neurodegenerative disorders, such as ones of these classes, are mediated by pathologies independent of an autoimmune reaction against myelin or, more generally an autoimmune reaction against any self-antigen. Some neurodegenerative disorders can be mediated in part by trinucleotide expansion in genes, resulting in proteins that cause polyglutamine aggregation. Examples of such disorders include: Huntington's disease, Spinalbulbar Muscular Atrophy (SBNIA or Kennedy's Disease) Dentatontbropallidoluysian Atrophy (DRPLA), Spinocerebellar Ataxia 1 (SCA1), Spinocerebellar Ataxia 2 (SCA2), Machado-Joseph Disease (MJD; SCA3), Spinocerebellar Ataxia 6 (SCA6), Spinocerebellar Ataxia 7 (SCA7), and Spinocerebellar Ataxia 12 (SCA 12).
Examples of neurodegenerative disorders mediated by prions include Creutzfeldt-Jakob disease (CJD) and so-called “mad-cow disease.” In another aspect, the disclosure features a method of evaluating a subject. The method includes detecting a TWEAK or TWEAK receptor (e.g., Fn14) protein or a nucleic acid encoding TWEAK or a TWEAK receptor in a subject. In one embodiment, the method includes correlating the result of the detection with the subject's risk for a neurological disorder, other injury, or pathology. The term “correlating” refers to describing the relationship between the presence or level of TWEAK or TWEAK-R protein or nucleic acid, and the presence or level of risk for a neurological disorder, other injury, or pathology. For example, increased expression can indicate that the subject has suffered a neuronal or other injury to the brain or spinal cord, or a neurological disorder, or is at risk for one. Such correlation may be displayed in a record, e.g., a printed or computer readable material, e.g., an informational, diagnostic, or instructional material, e.g., to the subject, health care provider, or insurance company, identifying the presence or level of TWEAK or a TWEAK-R protein or nucleic acid as a risk or diagnostic factor for a neurological, disorder, other injury, or pathology. In another embodiment, increased expression can indicate that the subject has had a neuronal or other injury or pathology in the brain or spinal cord or a neurological disorder. In one embodiment, a labeled agent that binds to TWEAK or a TWEAK receptor is administered to the subject and the subject is monitored (e.g., scanned) to detect one or more locations in the brain or spinal cord where TWEAK or a TWEAK receptor expressed. The method can identify locations where TWEAK or a TWEAK receptor expression is increased. In other embodiments, TWEAK or TWEAK receptor expression is detected in a biological sample from the subject.
In another aspect the disclosure features a method of evaluating a subject or performing a risk assessment for a neuronal disorder such as a mechanical neuronal trauma, a neurodegenerative disorder, or other disorder described herein. The method includes evaluating TWEAK or TWEAK-R protein or a nucleic acid encoding TWEAK or a TWEAK receptor in the subject or in a sample obtained from the subject. For example, the step of evaluating includes evaluating expression or activity of a TWEAK or TWEAK-R protein or a nucleic acid encoding TWEAK or a TWEAK receptor (e.g., by qualitative or quantitative analysis of mRNA, cDNA, or protein), or evaluating one or more nucleotides in a nucleic acid (genomic, mRNA, or cDNA) encoding TWEAK or a TWEAK receptor. In one embodiment, the subject has sustained, is suspected of having sustained, or is known or suspected to have a predisposition for a neuronal disorder, such as a mechanical neuronal trauma, a neurodegenerative disorder, or other disorder described herein. In one embodiment, the method includes administering a labeled TWEAK or TWEAK receptor binding agent (e.g., an antibody) to a subject, and evaluating localization of the labeled binding agent in the subject, e.g., by imaging the subject (e.g., imaging at least a portion of the brain of the subject). For example, a NMR-detectable antibody to a TWEAK receptor can be used to identify Fn14 overexpressing cells at site of damage from a neuronal or other injury or pathology in the brain or spinal cord or a neurological disorder.
Results of the evaluating can be used to provide a risk or an assessment of status for sustaining a neuronal disorder such as a mechanical neuronal trauma, a neurodegenerative disorder, or other disorder described herein, e.g., by comparison to a reference, e.g., a reference value for a normal subject, a control subject, or a value determined, e.g., for a cohort of subjects.
The method can be used to evaluate a treatment for a neuronal disorder such as a mechanical neuronal trauma, a neurodegenerative disorder, or other disorder described herein. For example, the subject is receiving a treatment for a neuronal disorder, e.g., a disorder described herein (e.g., a treatment using a TWEAK/TWEAK-R blocking agent, or other treatment for the particular disorder). The subject can be evaluated before, during, or after receiving the treatment, e.g., multiple times during the course of treatment. A decrease in TWEAK and/or TWEAK receptor expression can indicate an improvement in the condition of the subject.
The method can be used to identify a subject for treatment for a neuronal disorder such as a mechanical neuronal trauma, a neurodegenerative disorder, or other disorder described herein. The subject can be identified as a subject suited for such treatment as a function of results of the evaluating, e.g., the results show similarity to, e.g., statistically significant similarity to, a reference value indicative of a subject requiring such a treatment. For example, elevated TWEAK or TWEAK receptor expression can be indicative of a subject who can be treated with a TWEAK/TWEAK-R blocking agent or other treatment appropriate for the neuronal disorder.
The method can also be used to select a patient population for treatment. Expression of TWEAK or a TWEAK receptor is evaluated for one or more subjects. A set of one or more subjects who have elevated expression of TWEAK or a TWEAK receptor relative to a reference are selected. The subjects of the set are administered an agent that blocks a TWEAK/TWEAK-R interaction or activity or other treatment for a neuronal disorder such as a mechanical neuronal trauma, a neurodegenerative disorder, or other disorder described herein.
In another aspect, this disclosure features a method that includes a) determining the identity of at least one nucleotide in the TWEAK and/or a TWEAK receptor locus of a subject; and b) creating a record which includes information about the identity of the nucleotide and information relating to a parameter of the subject that corresponds to a neuronal disorder such as a mechanical neuronal trauma, a neurodegenerative disorder, or other disorder described herein, wherein the parameter is other than the genotype of TWEAK or TWEAK-R genes. The method can be used e.g., for gathering genetic information. In one embodiment, the determining includes evaluating a sample including human genetic material from the subject. A related method includes: (a) evaluating a parameter of a TWEAK and/or TWEAK receptor molecule (a TWEAK/TWEAK-R parameter) from a mammalian subject; and (b) evaluating a parameter related to a neuronal disorder such as a mechanical neuronal trauma, a neurodegenerative disorder, or other disorder described herein of the subject wherein the parameter is other than the parameter of (a).
The methods can also include (c) recording information about the TWEAK/TWEAK-R parameter and information about the parameter related to a neuronal disorder, wherein the information about the parameter and information about the phenotypic trait are associated with each other in a record, e.g., a database. For example, the parameter is a phenotypic trait of the subject, e.g., a parameter related to the disorder, e.g., a description of symptoms of the disorder, e.g., a qualitative or quantitative evaluation of such symptoms.
In one embodiment, the TWEAK and/or TWEAK receptor molecule is a polypeptide and the TWEAK/TWEAK-R parameter includes information about a TWEAK/TWEAK-R polypeptide. In another embodiment, the TWEAK and/or TWEAK receptor molecule is a nucleic acid that encodes TWEAK or TWEAK receptor and the TWEAK/TWEAK parameter includes information about the identity of a nucleotide in the TWEAK/TWEAK-R gene. Other parameters can relate to TWEAK and/or TWEAK receptor expression, activity, modification, or localization (e.g., subcellular or organismal).
In an embodiment, the subject is an embryo, blastocyst, or fetus. In another embodiment, the subject is a post-natal human, e.g., a child or an adult (e.g., at least 20, 30, 40, 50, 60, or 70 years of age).
In one embodiment, step (b) is performed before or concurrent with step (a). In one embodiment, the human genetic material includes DNA and/or RNA.
The method can further include comparing the TWEAK/TWEAK-R parameter to reference information, e.g., information about a corresponding nucleotide from a reference sequence. The method can be used, e.g., to provide a diagnosis of a neuronal disorder for a subject who does not yet exhibit symptoms or who does exhibit symptoms, but whose diagnosis is uncertain. In one embodiment, the reference sequence is from a reference subject that has a neuronal disorder such as a mechanical neuronal trauma, a neurodegenerative disorder, or other disorder described herein, e.g., a neurological disorder at an age associated with early onset for the disorder, e.g., in some cases an age of less than age 60, 55, 50, or 45.
In one embodiment, the method further includes comparing the nucleotide to a corresponding nucleotide from a genetic relative or family member (e.g., a parent, grandparent, sibling, progeny, prospective spouse, etc.).
In one embodiment, the method further includes evaluating risk or determining diagnosis of a neuronal disorder such as a mechanical neuronal trauma, a neurodegenerative disorder, or other disorder described herein in the subject as a function of the genotype.
In one embodiment, the method further includes recording information about the TWEAK/TWEAK-R parameter and a parameter related to a neuronal disorder such as a mechanical neuronal trauma, a neurodegenerative disorder, or other disorder described herein, e.g., in a record or database. For example, the information is recorded in linked fields of a database (e.g., TWEAK/TWEAK-R parameter is linked to at least one of: corresponding TWEAK/TWEAK-R parameter and/or data regarding comparison with the reference sequence). The nucleotide can be located in an exon, intron, or regulatory region of the TWEAK/TWEAK-R gene. For example, the nucleotide is a SNP. In one embodiment, a plurality of nucleotides (e.g., at least 10, 20, 50, 100, 500, or 1000 nucleotides (e.g., consecutive or non-consecutive)) in the TWEAK/TWEAK-R locus are evaluated. In another embodiment, a single nucleotide is evaluated.
In one embodiment, the method includes one or more of: evaluating a nucleotide position in the TWEAK/TWEAK-R locus on both chromosomes of the subject; recording the information (e.g., as phased or unphased information); aligning the genotyped nucleotides of the sample and the reference sequence; and identifying nucleotides that differ between the subject nucleotides and the reference sequence.
The method can be repeated for a plurality of subjects (e.g., at least 10, 25, 50, 100, 250, or 500 subjects).
In one embodiment, the method can include comparing the information of step a) and step b) to information in a database, and evaluating the association of the genotyped nucleotide(s) with a neuronal disorder such as a mechanical neuronal trauma, a neurodegenerative disorder, or other disorder described herein.
In another aspect, the invention features a computer readable record encoded with (a) a subject identifier, e.g., a patient identifier, (b) one or more results from an evaluation of the subject, e.g., a diagnostic evaluation described herein, e.g., the level of expression, level or activity of TWEAK or a TWEAK receptor, in the subject, and optionally (c) a value for or related to a neuronal disorder such as a mechanical neuronal trauma, a neurodegenerative disorder, or other disorder described herein, e.g., a value correlated with injury or disorder status or risk with regard to such injury or disorder. In one embodiment, the invention features a computer medium having a plurality of digitally encoded data records. Each data record includes a value representing the expression, level or activity of TWEAK or a TWEAK receptor levels or activity, in a sample or subject, and a descriptor of the sample or the subject. The descriptor of the sample can be an identifier of the sample, a subject from which the sample was derived (e.g., a patient), a diagnosis, or a treatment (e.g., a treatment described herein). In a preferred embodiment, the data record further includes values representing the level or location of expression, level or activity of genes other than TWEAK or TWEAK receptor (e.g., other genes associated with a neuronal disorder such as a mechanical neuronal trauma, a neurodegenerative disorder, or other disorder described herein, or other genes on an array). The data record can be structured as a table, e.g., a table that is part of a database such as a relational database (e.g., a SQL database of the Oracle or Sybase database environments). It is also possible to communicate information about a subject, e.g., by transmitting information, e.g., transmitting a computer readable record described herein, e.g., over a computer network.
All cited patents, patent applications, and references are hereby incorporated by reference in their entireties. In the case of conflict, the present application controls.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.



DESCRIPTION OF DRAWINGS
FIG. 1 is a bar graph showing TWEAK and Fn14 levels in FALS.
FIG. 2 is a bar graph showing that Fn14 wild type (WT) and knock out (KO) mice have similar numbers of TH+ neurons and a plot showing that Fn14 knock out mice have reduced TH+ neuron loss following MPTP treatment relative to wild type.
FIG. 3 is a plot showing that Fn14 deficiency reduces microglial activation following MPTP treatment. WT=wild type; Fn14=Fn14 deficient.



DETAILED DESCRIPTION
Administration of a TWEAK/TWEAK-R blocking agent, e.g., a TWEAK antibody, can reduce neuronal injury or pathology in the central or peripheral nervous system. A TWEAK/TWEAK-R blocking agent may also minimize damage associated with apoptosis, inflammation, or other secondary processes associated with a disorder described herein. As further described herein, a TWEAK/TWEAK-R blocking agent can be used to treat or prevent neurodegenerative diseases, e.g., disorders characterized by the degeneration (frequently gradual) and death of neurons, such as motor neurons, inter-neurons, or sensory neurons, as well as other neurological diseases.
Surprisingly, a TWEAK/TWEAK-R blocking agent can mitigate neuronal damage associated with a neurodegenerative disorder in which autoimmunity is not a principal mediator. The ability of the blocking agent to prevent neuronal cell death or to reduce inflammation (e.g., inflammation mediated by glial cells and astrocytes) may contribute to such therapeutic results.
In addition to neurodegenerative disorders in which autoimmunity is not a principal mediator, a TWEAK/TWEAK-R blocking agent can also be used to treat autoimmune disorders which cause neuronal damage. For example, the blocking agent can be used to treat multiple sclerosis, a condition in which the immune system attacks myelin sheaths of neurons. However, the blocking agent is also effective at treating other neuronal disorders in which autoimmunity may be implicated, e.g., ALS, in which the immune system may attack motor neurons themselves, rather than the surrounding myelin tissue. Accordingly, a TWEAK/TWEAK-R blocking agent can be used to treat autoimmune disorders with a neuronal pathology that are not based on immunological damage to myelin.
The term “treating” refers to administering a therapy in an amount, manner, and/or mode effective to improve or prevent a condition, symptom, or parameter associated with a disorder (e.g., SCI, TBI, ALS, Parkinson's Disease, Huntington's Disease, Alzheimer's Disease, or other disorder described herein) or to prevent onset, progression, or exacerbation of the disorder (including secondary damage caused by the disorder, e.g., neuronal injury to or inflammation in the brain, spinal cord, or peripheral nervous system), to either a statistically significant degree or to a degree detectable to one skilled in the art. Accordingly, treating can achieve therapeutic and/or prophylactic benefits. An effective amount, manner, or mode can vary depending on the subject and may be tailored to the subject.
TWEAK/TWEAK-Receptor Blocking Agents
A variety of agents can be used as a TWEAK/TWEAK-R blocking agent to treat neuronal or other injury or pathology in the brain or spinal cord or neurological disorders. The agent may be any type of compound (e.g., small organic or inorganic molecule, nucleic acid, protein, or peptide mimetic) that can be administered to a subject. In one embodiment, the blocking agent is a biologic, e.g., a protein having a molecular weight of between 5-300 kDa. For example, a TWEAK/TWEAK-R blocking agent may inhibit binding of TWEAK to a TWEAK receptor or may prevent TWEAK-mediated NF-κB activation. A typical TWEAK/TWEAK-R blocking agent can bind to TWEAK or a TWEAK receptor, e.g., Fn14. A TWEAK/TWEAK-R blocking agent that binds to TWEAK may alter the conformation of TWEAK or a TWEAK receptor, block the binding site on TWEAK or a TWEAK receptor, or otherwise decrease the affinity of TWEAK for a TWEAK receptor or prevent the interaction between TWEAK and a TWEAK receptor. A TWEAK/TWEAK-R blocking agent (e.g., an antibody) may bind to TWEAK or to a TWEAK receptor with a Kd of less than 10−6, 10−7, 10−8, 10−9, or 10−10 M. In one embodiment, the blocking agent binds to TWEAK with an affinity at least 5, 10, 20, 50, 100, 200, 500, or 1000 better than its affinity for TNF or another TNF superfamily member (other than TWEAK). In one embodiment, the blocking agent binds to the TWEAK receptor with an affinity at least 5, 10, 20, 50, 100, 200, 500, or 1000-fold better than its affinity for the TNF receptor or a receptor for another TNF superfamily member. A preferred TWEAK/TWEAK-R blocking agent specifically binds TWEAK or a TWEAK receptor, such as a TWEAK or TWEAK-R specific antibody.
Exemplary TWEAK protein molecules include human TWEAK (e.g., AAC51923, shown as SEQ JD NO:1)), mouse TWEAK (e.g., NP_035744.1), rat TWEAK (e.g., XP_340827.1), and Pan troglodytes TWEAK (e.g., XP_511964.1). Also included are proteins that include an amino acid sequence at least 90, 92, 95, 97, 98, 99% identical and completely identical to the mature processed region of the aforementioned TWEAK proteins (e.g., an amino acid sequence at least 90, 92, 95, 97, 98, 99% identical or completely identical to amino acids X1-249 of SEQ ID NO: 1, where amino acid X1 is selected from the group of residues 75-115 of SEQ ID NO:1, e.g., X1 is residue Arg 93 of SEQ ID NO:1) and proteins encoded by a nucleic acid that hybridizes under high stringency conditions to a human, mouse, rat, or Pan troglodytes gene encoding a naturally occurring TWEAK protein. Preferably, a TWEAK protein, in its processed mature form, is capable of providing at least one TWEAK activity, e.g., ability to activate Fn14.
Exemplary TWEAK receptor molecules include Fn14. Exemplary Fn14 protein molecules include human Fn14 (e.g., NP_057723.1, shown as SEQ ID NO:2), mouse Fn14 (e.g., NP_038777.1), and rat Fn14 (e.g., NP_851600.1), as well as soluble proteins that include an amino acid sequence at least 90, 92, 95, 97, 98, 99% identical or 100% identical to the extracellular domain of Fn14 (and TWEAK-binding fragments thereof) and proteins encoded by a nucleic acid that hybridizes under high stringency conditions to a human, mouse, rat, or Pan troglodytes gene encoding a naturally-occurring Fn14 protein. Preferably, an Fn14 protein useful in the methods described herein is a soluble Fn14 (lacking a transmembrane domain) that includes a region that binds to a TWEAK protein, e.g., an amino acid sequence at least 90, 92, 95, 97, 98, or 99% identical, or completely identical, to amino acids 28-X1 of SEQ ID NO:2, where amino acid X1 is selected from the group of residues 68 to 80 of SEQ ID NO:2.
Calculations of “homology” or “sequence identity” between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossom 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.
As used herein, the term “hybridizes under high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, Aqueous and nonaqueous methods are described in that reference and either can be used High stringency hybridization conditions include hybridization in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C., or substantially similar conditions.
Exemplary TWEAK/TWEAK-R blocking agents include antibodies that bind to TWEAK or a TWEAK receptor and soluble forms of the TWEAK receptor that compete with cell surface TWEAK receptor for binding to TWEAK. An example of a soluble form of the TWEAK receptor is an Fc fusion protein that includes at least a portion of the extracellular domain of TWEAK receptor (e.g., a soluble TWEAK binding fragment of TWEAK receptor), referred to as TWEAK-R-Fc. Other soluble forms of a TWEAK receptor, e.g., forms that do not include an Fc domain, can also be used. Antibody blocking agents are further discussed below. Other types of blocking agents, e.g., small molecules, nucleic acid or nucleic acid-based aptamers, and peptides, can be isolated by screening, e.g., as described in Jhaveri et al. (2000) Nat. Biotechnol, 18:1293 and U.S. Pat. No. 5,223,409. Exemplary assays for determining if an agent binds to TWEAK or TWEAK receptor and for determining if an agent modulates a TWEAK/TWEAK-R interaction are described, e.g., in U.S. Pub. App. No. 2004-0033225.
An exemplary soluble form of the TWEAK-R protein includes a region of the TWEAK-R protein that binds to TWEAK, e.g., about amino acids 32-75, 31-75, 31-78, or 28-79 of SEQ ID NO:2. This region can be physically associated, e.g., fused to another amino acid sequence, e.g., an Fc domain, at its N- or C-terminus. The region from TWEAK receptor can be spaced by a linker from the heterologous amino acid sequence. U.S. Pat. No. 6,824,773 describes an exemplary TWEAK receptor fusion protein.
Antibodies
Exemplary TWEAK/TWEAK-R blocking agents include antibodies that bind to TWEAK and/or a TWEAK receptor. In one embodiment, the antibody inhibits the interaction between TWEAK and a TWEAK receptor, e.g., by physically blocking the interaction, decreasing the affinity of TWEAK and/or a TWEAK receptor for its counterpart, disrupting or destabilizing TWEAK complexes, sequestering TWEAK or a TWEAK receptor, or targeting TWEAK or a TWEAK receptor for degradation. In one embodiment, the antibody can bind to TWEAK or a TWEAK receptor at one or more amino acid residues that participate in the TWEAK/TWEAK-R binding interface. Such amino acid residues can be identified, e.g., by alanine scanning. In another embodiment, the antibody can bind to residues that do not participate in the TWEAK/TWEAK-R binding. For example, the antibody can alter a conformation of TWEAK or a TWEAK receptor and thereby reduce binding affinity, or the antibody may sterically hinder TWEAK/TWEAK-R binding. In one embodiment, the antibody can prevent activation of a TWEAK/TWEAK-R mediated event or activity (e.g., NF-κB activation).
As used herein, the term “antibody” refers to a protein that includes at least one immunoglobulin variable region, e.g., an amino acid sequence that provides an immunoglobulin variable domain or an immunoglobulin variable domain sequence. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term “antibody” encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab fragments, F(ab′)2 fragments, Fd fragments, Fv fragments, and dAb fragments) as well as complete antibodies, e.g., intact and/or full length immunoglobulins of types IgA, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgE, IgD, IgM (as well as subtypes thereof). The light chains of the immunoglobulin may be of types kappa or lambda. In one embodiment, the antibody is glycosylated. An antibody can be functional for antibody-dependent cytotoxicity and/or complement-mediated cytotoxicity, or may be non-functional for one or both of these activities.
The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (FR). The extent of the FR's and CDR's has been precisely defined (see, Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; and Chothia. C. et al (1987) J. Mol. Biol. 196:901-917). Kabat definitions are used herein. Each VH and VL is typically composed of three CDR's and four FR's, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
An “immunoglobulin domain” refers to a domain from the variable or constant domain of immunoglobulin molecules. Immunoglobulin domains typically contain two β-sheets formed of about seven β-strands, and a conserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay (1988) Ann. Rev. Immunol. 6:381-405). An “immunoglobulin variable domain sequence” refers to an amino acid sequence that can form a structure sufficient to position CDR sequences in a conformation suitable for antigen binding. For example, the sequence may include all or part of the amino acid sequence of a naturally-occurring variable domain. For example, the sequence may omit one, two, or more N- or C-terminal amino acids, internal amino acids, may include one or more insertions or additional terminal amino acids, or may include other alterations. In one embodiment, a polypeptide that includes an immunoglobulin variable domain sequence can associate with another immunoglobulin variable domain sequence to form a target binding structure (or “antigen binding site”), e.g., a structure that interacts with TWEAK or a TWEAK receptor.
The VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy immunoglobulin chain (HC) or light immunoglobulin chain (LC), respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains. The heavy and light immunoglobulin chains can be connected by disulfide bonds. The heavy chain constant region typically includes three constant domains, CH1, CH2, and CH3. The light chain constant region typically includes a CL domain. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
One or more regions of an antibody can be human, effectively human, or humanized. For example, one or more of the variable regions can be human or effectively human. For example, one or more of the CDRs, e.g., HC CDR1, EC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3, can be human. Each of the light chain CDRs can be human. HC CDR3 can be human. One or more of the framework regions can be human, e.g., FR1, FR2, FR3, and FR4 of the HC or LC. In one embodiment, all the framework regions are human, e.g., derived from a human somatic cell, e.g., a hematopoietic cell that produces immunoglobulins, or a non-hematopoietic cell. In one embodiment, the human sequences are germline sequences, e.g., encoded by a germline nucleic acid. One or more of the constant regions can be human, effectively human, or humanized. In another embodiment, at least 70, 75, 80, 85, 90, 92, 95, or 98% of the framework regions (e.g., FR1, FR2, and FR3, collectively, or FR1, FR2, FR3, and FR4, collectively) or the entire antibody can be human, effectively human, or humanized. For example, FR1, FR2, and FR3 collectively can be at least 70, 75, 80, 85, 90, 92, 95, 98, or 99% identical, or completely identical, to a human sequence encoded by a human germline segment.
An “effectively human” immunoglobulin variable region is an immunoglobulin variable region that includes a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. An “effectively human” antibody is an antibody that includes a sufficient number of human amino acid positions such that the antibody does not elicit an immunogenic response in a normal human.
A “humanized” immunoglobulin variable region is an immunoglobulin variable region that is modified such that the modified form elicits less of an immune response in a human than does the non-modified form, e.g., is modified to include a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. Descriptions of “humanized” immunoglobulins include, for example, U.S. Pat. Nos. 6,407,213 and 5,693,762. In some cases, humanized immunoglobulins can include a non-human amino acid at one or more framework amino acid positions.
Antibody Generation
Antibodies that bind to TWEAK or a TWEAK receptor can be generated by a variety of means, including immunization, e.g., using an animal, or in vitro methods such as phage display. All or part of TWEAK or a TWEAK receptor can be used as an immunogen or as a target for selection. For example, TWEAK or a fragment thereof, or a TWEAK receptor or a fragment thereof, can be used as an immunogen. In one embodiment, the immunized animal contains immunoglobulin-producing cells with natural, human, or partially human immunoglobulin loci. In one embodiment, the non-human animal includes at least a part of a human immunoglobulin gene. For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci. Using the hybridoma technology, antigen-specific monoclonal antibodies derived from the genes with the desired specificity may be produced and selected. See, e.g., XENOMOUSETM, Green at al. (1994) Nat. Gen, 713-21; U.S. Pub. App. No. 2003-0070185; U.S. Pat. No. 5,789,650; and PCT Pub. No. WO 96/34096.
Non-human antibodies to TWEAK or a TWEAK receptor can also be produced, e.g., in a rodent. The non-human antibody can be humanized, e.g., as described in EP 239 400; U.S. Pat. Nos. 6,602,503; 5,693,761; and 6,407,213, deimmunized, or otherwise modified to make it effectively human.
EP 239 400 (Winter et al.) describes altering antibodies by substitution (within a given variable region) of their complementarity determining regions (CDRs) for one species with those from another. Typically, CDRs of a non-human (e.g., murine) antibody are substituted into the corresponding regions in a human antibody by using recombinant nucleic acid technology to produce sequences encoding the desired substituted antibody. Human constant region gene segments of the desired isotype (usually gamma I for CH and kappa for CL) can be added and the humanized heavy and light chain genes can be co-expressed in mammalian cells to produce soluble humanized antibody. Other methods for humanizing antibodies can also be used. For example, other methods can account for the three-dimensional structure of the antibody, framework positions that are in three-dimensional proximity to binding determinants, and immunogenic peptide sequences. See, e.g., PCT Pub. No. WO 90/07861; U.S. Pat. Nos. 5,693,762; 5,693,761; 5,585,089; and 5,530,101; Tempest et al. (1991) Biotechnology 9:266-271 and U.S. Pat. No. 6,407,213.
Fully human monoclonal antibodies that bind to TWEAK or a TWEAK receptor can be produced, e.g., using in vitro-primed human splenocytes, as described by Boerner et al. (1991) J. Immunol. 147:86-95. They may be prepared by repertoire cloning as described by Persson et al. (1991) Proc. Natl. Acad. Sci. USA 88:2432-2436 or by Huang and Stollar (1991) J. Immunol. Methods 141:227-236; also U.S. Pat. No. 5,798,230. Large nonimmunized human phage display libraries may also be used to isolate high affinity antibodies that can be developed as human therapeutics using standard phage technology (see, Hoogenboom et al. (1998) Immunotechnology 4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-378; and U.S. Pub. App. No. 2003-0232333).
Antibody and Protein Production
Antibodies and other proteins described herein can be produced in prokaryotic and eukaryotic cells. In one embodiment, the antibodies (e.g., scFv's) are expressed in a yeast cell such as Pichia (see, e.g., Powers et al. (2001) J. Immunol. Methods 251:123-35), Hanseula, or Sacchamtnyces. 
Antibodies, particularly full length antibodies, e.g., IgG's, can be produced in mammalian cells. Exemplary mammalian host cells for recombinant expression include Chinese Hamster Ovary (CHO cells) (including dhfr− CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621), lymphocytic cell lines, e.g., NS0 myeloma cells and SP2 cells, COS cells, K562, and a cell from a transgenic animal, e.g., a transgenic mammal. For example, the cell is a mammary epithelial cell.
In addition to the nucleic acid sequence encoding the immunoglobulin domain, the recombinant expression vectors may carry additional nucleic acid sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and 5,179,017). Exemplary selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr− host cells with methotrexate selectior/amplification) and the neo gene (for G418 selection).
In an exemplary system for recombinant expression of an antibody (e.g., a full length antibody or an antigen-binding portion thereat), a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr− CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus, and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40 enhancer/AcMLP promoter regulatory element) to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, to transfect the host cells, to select for transformants, to culture the host cells, and to recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G.
Antibodies (and Fc fusions) may also include modifications, e.g., modifications that alter Fc function, e.g., to decrease or remove interaction with an Fc receptor or with C1q, or both. For example, the human IgG1 constant region can be mutated at one or more residues, e.g., one or more of residues 234 and 237, e.g., according to the numbering in U.S. Pat. No. 5,648,260. Other exemplary modifications include those described in U.S. Pat. No. 5,648,260.
For some proteins that include an Fc domain, the antibody/protein production system may be designed to synthesize antibodies or other proteins in which the Fc region is glycosylated. For example, the Fc domain of IgG molecules is glycosylated at asparagine 297 in the CH2 domain. The Fc domain can also include other eukaryotic post-translational modifications. In other cases, the protein is produced in a form that is not glycosylated.
Antibodies and other proteins can also be produced by a transgenic animal. For example, U.S. Pat. No. 5,849,992 describes a method for expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter and nucleic acid sequences encoding the antibody of interest, e.g., an antibody described herein, and a signal sequence for secretion. The milk produced by females of such transgenic mammals includes, secreted-therein, the protein of interest, e.g., an antibody or Fc fusion protein. The protein can be purified from the milk, or for some applications, used directly.
Methods described in the context of antibodies can be adapted to other proteins, e.g., Fc fusions and soluble receptor fragments.
Nucleic Acid Blocking Agents
In certain implementations, nucleic acid blocking agents are used to decrease expression of an endogenous gene encoding TWEAK or a TWEAK receptor, e.g., Fn14. In one embodiment, the nucleic acid antagonist is an siRNA that targets mRNA encoding TWEAK or a TWEAK receptor. Other types of antagonistic nucleic acids can also be used, e.g., a dsRNA, a ribozyme, a triple-helix former, or an antisense nucleic acid.
siRNAs are small double stranded RNAs (dsRNAs) that optionally include overhangs. For example, the duplex region of an siRNA is about 18 to 25 nucleotides in length, e.g., about 19, 20, 21, 22, 23, or 24 nucleotides in length. Typically, the siRNA sequences are exactly complementary to the target mRNA. dsRNAs and siRNAs in particular can be used to silence gene expression in mammalian cells (e.g., human cells). See, e.g., Clemens et al. (2000) Proc. Natl. Acad. Sci. USA 97:6499-6503; Billy et al. (2001) Proc. Natl. Sci. USA 98:14428-44433; Elbashir et al. (2001) Nature 411:494-498; Yang et al. (2002)Proc. Natl. Acad. Sci. USA 99:99429947, U.S. Pub App. Nos. 2003-0166282; 2003-0143204: 2004-0038278; and 2003-0224432.
Anti-sense agents can include, for example, from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 nucleotides), e.g., about 8 to about 50 nucleobases, or about 12 to about 30 nucleobases. Anti-sense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic add and modulate its expression. Anti-sense compounds can include a stretch of at least eight consecutive nucleobases that are complementary to a sequence in the target gene. An oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted.
Hybridization of antisense oligonucleotides with mRNA (e.g., an mRNA encoding TWEAK or a TWEAK receptor) can interfere with one or more of the normal functions of mRNA. The functions of mRNA to be interfered with include all key functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA. Binding of specific protein(s) to the RNA may also be interfered with by antisense oligonucleotide hybridization to the RNA.
Exemplary antisense compounds include DNA or RNA sequences that specifically hybridize to the target nucleic acid, e.g., the mRNA encoding TWEAK or a TWEAK receptor. The complementary region can extend for between about 8 to about 80 nucleobases. The compounds can include one or more modified nucleobases. Modified nucleobases may include, e.g., 5-substituted pyrimidines such as 5-iodouracil, 5-iodocytosine, and C5-propynyl pyrimidines such as C5-propynylcytosine and C5-propynyluracil. Other suitable modified nucleobases include N4 (C1-C12) alkylaminocytosines and N4,N4—(C1-C12) dialkylaminocytosines. Modified nucleobases may also include 7-substituted-8-aza-7-deazapurines and 7-substituted-7-deazapurines such as, for example, 7-iodo-7-deazapurines, 7-cyano-7-deazapurines, 7-aminocarbonyl-7-deazapurines. Examples of these include 6-amino-7-iodo-7-deazapurines, 6-amino-7-cyano-7-deazapurines, 6-amino-7-aminocarbonyl-7-deazapurines, 2-amino-6-hydroxy-7-iodo-7-deazapurines, 2-amino-6-hydroxy-7-cyano-7-deazapurines, and 2-amino-6-hydroxy-7-aminocarbonyl-7-deazapurines. Furthermore, N6—(C1-C12) alkylaminopurines and N6,N6—(C1-C12) dialkylaminopurines, including N6-methylaminoadenine and N6,N6-dimethylaminoadenine, are also suitable modified nucleobases. Similarly, other 6-substituted purines including, for example, 6-thioguanine may constitute appropriate modified nucleobases. Other suitable nucleobases include 2-thiouracil, 8-bromoadenine, 8-bromoguanine, 2-fluoroadenine, and 2-fluorozuanine. Derivatives of any of the aforementioned modified nucleobases are also appropriate. Substituents of any of the preceding compounds may include C1-C30 alkyl, C2-C30 alkenyl, C2-C30 alkynyl, aryl, aralkyl, heteroaryl, halo, amino, amido, nitro, thio, sulfonyl, carboxyl, alkoxy, alkylcarbonyl, alkoxycarbonyl, and the like.
Descriptions of other types of nucleic acid agents are also available. See, e.g., U.S. Pat. Nos. 4,987,071; 5,116,742; and 5,093,246; Woolf et al. (1992) Proc. Natl. Acad. Sci. USA 89:7305-7309; Antisense RNA and DNA, D. A, Melton, Ed, Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988); Haseloff and Gerlach (1988) Nature 334:585-591; Helene, C. (1991) Anticancer Drug Des, 6:569-84; Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14:807-815.
The nucleic acids described herein, e.g., an anti-sense nucleic acid described herein, can be incorporated into a gene construct to be used as a part of a gene therapy protocol to deliver nucleic acids that can be used to express and produce agents, e.g., anti-sense nucleic acids, within cells. Expression constructs of such components may be administered in any biologically-effective cattier, e.g., any formulation or composition capable of effectively delivering the component gene to cells in vivo. Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, lentivirus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNA can be delivered with the help of for example, cationic liposomes lipofectin) or derivatized (e.g., antibody conjugated), polylysine conjugates, gramicidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO4 precipitation canned out in vivo.
A preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid, e.g., a cDNA. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken tip viral vector nucleic acid.
Retrovirus vectors and adeno-associated virus vectors can be used as a recombinant gene delivery system for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE, and pEM which are known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include *Crip, *Cre, *2, and *Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, for example, Eglitis et al, (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88; 8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88; 8377-8381; Chowdhury et. al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Pat. Nos. 4,868,116 and 4,980,286; PCT Pub. Nos. WO 89/07136, WO 89/02468, WO 89/05345, and WO 92/07573).
Another viral gene delivery system utilizes adenovirus-derived vectors. See, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252; 431-434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known to those skilled in the art.
Yet another viral vector system useful for delivery of the subject gene is the adeno-associated virus (AAV). Set, for example, Platte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973.
Artificial Transcription Factors
Artificial transcription factors can also be used to regulate expression of TWEAK and/or a TWEAK receptor. The artificial transcription factor can be designed or selected, from a library, e.g., for ability to bind to a sequence in an endogenous gene encoding TWEAK or a TWEAK receptor, e.g., in a regulatory region, e.g., the promoter. For example, the artificial transcription factor can be prepared by selection in vitro (e.g., using phage display, U.S. Pat. No. 6,534,261) or in vivo, or by design based on a recognition code (see, e.g., PCT Pub. No. WO 00/42219 and U.S. Pat. No. 6,511,808). See, e.g., Rebar et al. (1996) Methods Enzyme 267:129; Greisman and Pabo (1997) Science 275:657; Isalan et al. (2001) Nat. Biotechnol. 19:656; and Wu et al. (1995) Proc. Natl. Acad. Sci. USA 92:344 for, among other things, methods for creating libraries of varied zinc finger domains.
Optionally, an artificial transcription factor can be fused to a transcriptional regulatory domain, e.g., an activation domain to activate transcription or a repression domain to repress transcription. In particular, repression domains can be used to decrease expression of endogenous genes encoding TWEAK or TWEAK receptor. The artificial transcription factor can itself be encoded by a heterologous nucleic acid that is delivered to a cell or the protein itself can be delivered to a cell (see, e.g., U.S. Pat. No. 6,534,261). The heterologous nucleic acid that includes a sequence encoding the artificial transcription factor can be operably linked to an inducible promoter, e.g., to enable fine control of the level of the artificial transcription factor in the cell, e.g., a neuronal or glial cell, e.g., at or near a site of neuronal or other injury in the brain or spinal cord or at the site of neurodegeneration caused by a neurological disorder.
Spinal Cord Injury (SCI)
A TWEAK/TWEAK-R blocking agent can be administered to treat spinal cord injury (SCI). Spinal cord injury (SCI) occurs when an event, typically a traumatic event, results in damage to cells within the spinal cord or severs the nerve tracts that relay signals up and down the spinal cord. Common types of SCI include contusion (bruising of the spinal cord) and compression (caused by pressure on the spinal cord). Other types of injuries include lacerations (severing or tearing of some nerve fibers, such as damage caused by a gun shot wound), and central cord syndrome (specific damage to the corticospinal tracts of the cervical region of the spinal cord).
Severe SCI often causes paralysis (loss of control over voluntary movement and muscles of the body) and loss of sensation and reflex fruition below the point of injury, including autonomic activity such as breathing and other activities such as bowel and bladder control. Other symptoms such as pain or sensitivity to stimuli, muscle spasms, and sexual dysfunction may develop over time. SCI patients are also prone to develop secondary medical problems, such as bladder infections, lung infections, and bed sores.
While recent advances in emergency care and rehabilitation allow many SCI patients to survive, methods for reducing the extent of injury and for restoring function are still limited. Immediate treatment for acute SCI can include administration (local and/or systemic) of a TWEAK/TWEAK-R blocking agent. Immediate treatment can further include one or more of: techniques to relieve cord compression, drug therapy with corticosteroids such as methylprednisolone to minimize cell damage, and stabilization of the vertebrae of the spine to prevent further injury. One or more of these treatments can be used in combination with a TWEAK/TWEAK-R blocking agent. These immediate treatments can be provided promptly, e.g., within 2, 4, 8, 12, or 24 hours of injury.
Administration of a TWEAK/TWEAK-K blocking agent can also be used as a rehabilitation strategy, e.g., to minimize long-term disability or otherwise improve a condition of the subject, e.g., ameliorating one or more SCI symptoms. A neurological exam can be performed to determine the location (e.g., which spinal region (e.g., cervical, thoracic, lumbar, sacral) and which spinal levels (e.g., C1-C7, T1-T12, T12-L5, S1-S5)) have been injured. The extent and progression of, and improvement in, the injury can be measured by various criteria, e.g., the American Spinal Injury Association uses a scale of 0-5 to assess motor strength in SCI: 0— no contraction or movement; 1—minimal movement; 2—active movement, but not against gravity; 3—active movement against gravity; 4—active movement against resistance; 5—active movement against full resistance.
The efficacy and dosing of a TWEAK/TWEAK-R blocking agent can be evaluated in an animal model of SCI. Examples include: a laminectomy, e.g., at the 9th-to-10th thoracic (T9-T10) spinal vertebrae, followed by a lateral hemisection at the T9-T10 level (Teng et. al. (2002) Proc. Natl. Acad, Sci. USA 99:3024-3029); a crushing model (see, e.g., Wamil et al. (1998) Proc. Natl. Acad. Sci. USA 95:13188-13193); and a contusive model (Kuhn and Wrathall (1998) J. Neurotrauma 15:125-40).
Traumatic Brain Injury (TBI)
A TWEAK/TWEAK-R blocking agent can be administered to treat traumatic brain injury (TBI). TBI is typically the result of physical damage to the head. For example, TBI occurs when a sudden physical assault on the head causes damage to the brain. The damage can be focal, confined to one area of the brain, or diffuse, involving more than one area of the brain. TBI can result from a closed head injury or a penetrating head injury. A closed head injury occurs when the head suddenly and violently hits an object, but the object does not break through the skull. A penetrating head injury occurs when an object pierces the skull and enters the brain tissue. Several types of traumatic injuries can affect the head and brain. A skull fracture occurs when the bone of the skull cracks or breaks. A depressed skull fracture occurs when pieces of the broken skull press into the tissue of the brain. This can cause bruising of the brain tissue, called a contusion. A contusion can also occur in response to shaking of the brain within the confines of the skull, an injury called “countrecoup.” Shaken Baby Syndrome is a severe form of head injury that occurs when a baby is shaken forcibly enough to cause extreme countrecoup injury. Damage to a major blood vessel within the head can cause a hematoma or heavy bleeding into or around the brain.
Symptoms of a TBI may include headache, nausea, confusion or other cognitive problems, a change in personality, depression, irritability, and other emotional and behavioral problems. Some people may have seizures or enter a coma as a result of a TBI.
Immediate treatment for TBI can include administration (local and/or systemic) of a TWEAK/TWEAK-R blocking agent. Other immediate treatment can include surgery to control bleeding in and around the brain, monitoring and controlling intracranial pressure, insuring adequate blood flow to the brain, and treating the body for other injuries and infection. One or more of these treatments can be used in combination with a TWEAK/TWEAK-R blocking agent. Immediate treatments can be provided promptly; e.g., within 2, 4, 8, 12 or 24 hours of injury.
Later treatment can also include administration (local and/or systemic) of a TWEAK/TWEAK-R blocking agent, e.g., to improve the condition of the subject. The Glasgow Coma Scale can be used to rate the extent of injury and chances of recovery. The scale (3-15) involves testing for three patient responses: eye opening, best verbal response, and best motor response. A high score indicates a good prognosis and a low score indicates a poor prognosis. A TWEAK/TWEAK-R blocking agent can be used to ameliorate a subject's performance on this scale, or other assessment criteria.
The efficacy and dosing of a TWEAK/TWEAK-R blocking agent can be evaluated in an animal model of TBI, e.g., a mouse model (see, e.g., Zohar et (2003) Neuroscience 118:949-955).
Amyotrophic Lateral Sclerosis (ALS)
A TWEAK/TWEAK-R blocking agent can be administered to treat amyotrophic lateral sclerosis (ALS). ALS, sometimes called Lou Gehrig's disease, is a rapidly progressive neurological disease that attacks the nerve cells (neurons) that control voluntary muscles. The disease is one of many motor neuron diseases characterized by the gradual degeneration and death of motor neurons.
In ALS, both the upper motor neurons (those in the bran and the lower motor neurons (those in the spinal cord) degenerate or die. The cessation of signals to the muscles causes than to gradually weaken, atrophy, and twitch (e.g., experience fasoiculations). Eventually, the ability of the brain to start and control voluntary movement is lost.
ALS causes weakness with a wide range of disabilities. Eventually, all muscles under voluntary control are affected, and patients lose their strength and the ability to move their arms, legs, and body. When muscles in the diaphragm and chest wall fail, patients lose the ability to breathe without ventilatory support. Most people with ALS die from respiratory failure, usually within 3 to 5 years from the onset of symptoms. However, about 10 percent of ALS patients survive for 10 or more years.
A TWEAK/TWEAK-R blocking agent can be used to delay onset, reduce severity, and reduce the rate of progression of ALS. The agent can be used to treat long-term survivors as well patients who exhibit more rapid disease progression. The severity and progression of and improvement in, ALS can be monitored by assessment criteria, e.g., the Appel ALS Ratine. Scale (AALS). It includes assessment of ten parameters (Speech; Salivation; Swallowing; Handwriting; Cutting Food and Handling Utensils; Dressing and Hygiene; Tinning in Bed and Adjusting Bed Clothes; Walking; Climbing Stairs; Breathing); within each parameter, a score ranging from 0 (poor function or inability to perform the function) to 4 (normal function) is assigned. This rating scale provides a single quantitative index of clinical status that documents the variable manifestation and progression of ALS. An increase in the score indicates an improvement in the patient's condition.
As many as 20,000 Americans have ALS, and an estimated 5,000 people in the United States are diagnosed with the disease each years ALS is one of the most common neuromuscular diseases worldwide, and people of all races and ethnic backgrounds are affected. ALS most commonly strikes people between 40 and 60 years of age, but younger and older people also can develop the disease. Men are affected more often than women.
In 90 to 95 percent of all ALS cases, the disease occurs apparently at random with no clearly associated risk factors. About 5 to 10 percent of all ALS cases are inherited. The familial form of ALS (FALS) usually results from a pattern of inheritance that requires only one parent to carry the gene responsible for the disease. About 20 percent of all FALS cases result from a specific genetic defect that leads to mutation of the enzyme known as superoxide dismutase 1 (SOD1). PALS can also be caused by other genetic mutations. In some implementations, subjects who are the progeny of a FALS patient or of a FALS carrier, or who are identified as having PALS (e.g., by a genetic test), are given a prophylactic dose of a TWEAK/TWEAK-R blocking agent, e.g., prior to showing physical symptoms of ALS.
The onset of ALS may be subtle and gradual. The earliest symptoms may include twitching, cramping, or stiffness of muscles; muscle weakness affecting an arm or a leg; slurred and nasal speech; or difficulty chewing or swallowing. These general complaints then develop into more obvious weakness or atrophy that may cause a physician to suspect ALS.
Regardless of the part of the body first affected by the disease, muscle weakness and atrophy spread to other parts of the body as the disease progresses. Patients have increasing problems with moving, swallowing (dysphagia), and speaking or forming words (dysarthria). Symptoms of upper motor neuron involvement include tight and stiff muscles (spasticity) and exaggerated reflexes (hyperreflexia) including an overactive gag reflex. An abnormal reflex commonly called Babinski's sign (the large toe extends upward as the sole of the foot is stimulated in a certain way) also indicates upper motor neuron damage. Symptoms of lower motor neuron degeneration include muscle weakness and atrophy, muscle cramps, and fleeting twitches of muscles that can be seen under the skin. A TWEAK/TWEAK-R blocking agent can be used to ameliorate one or more these symptoms, or to reduce the rate of disease progression.
To be diagnosed with ALS, patients generally must have signs and symptoms of both upper and lower motor neuron damage not attributed to other casues.
Riluzole (RILUTEK®) has been approved for treating ALS, Riluzole is believed to reduce damage to motor neurons by decreasing the release of glutamate. Clinical trials with ALS patients showed that riluzole prolongs survival by several months, mainly in those with difficulty swallowing. Riluzole can be used in combination with a TWEAK/TWEAK-R blocking agent.
The efficacy and dosing of a TWEAK/TWEAK-R blocking agent can be evaluated in an animal model of ALS, e.g., in a mouse or rat overexpressing a mutant form of the human Cu, Zn superoxide dismutase gene (SOD1). In seine models, the mutant form carries a G93A mutation in SOD1 (see, e.g., Tu et al. (1996) Proc. Natl. Acad. Set. USA 93:3155-3160; Grieb (2004) Folia Neuropathol. 42:239-48; Storkebaum et al. (2005) Nat. Neurosci. 3:85-92; Sharp et al. (2005) Neuroscience 130:897-910). Transgenic rodents expressing human mutated (inn) SOD-1 develop relatively selective and fatal degeneration of motoneurons. Other transgenic mouse models carry a G85R mutation found in FALS; these mice develop motor neuron disease at 8-10 months of age (Amendola et al. (2004) Eur J. Neurosci. 20:2822-2826).
Parkinson's Disease
A TWEAK/TWEAK-R blocking agent can be administered to treat Parkinson's Disease. Parkinson's Disease is a slowly progressive neurodegenerative disorder caused by damaged or dead dopamine neurons in the substantia nigra, a region of the brain that controls balance and coordinates muscle movement.
A TWEAK/TWEAK-R blocking agent can be used to delay onset, reduce severity, and reduce the rate of progression of Parkinson's Disease. The agent can be used to treat long-term survivors as well as patients who exhibit more rapid disease progression.
Four symptoms of Parkinson's Disease often appear gradually but increase in severity with time. They are: trembling in hands, arms, legs, jaw, and face; rigidity or stiffness of the limbs and trunk; slowness of motor movements; postural instability or impaired balance and coordination. A TWEAK/TWEAK-R blocking agent can be used to ameliorate one or more these symptoms, or to reduce the rate of disease progression. The severity and progression of and improvement in, Parkinson's Disease can be assessed by a number of criteria including Hoehn and Yahr Staging of Parkinson's Disease (which classifies the disease into one of five levels with 5 being the most advanced disease); the Schwab and England Activities of Daily Living assessment (which classifies the disease as a percentage 0% (vegetative state) to 100% (completely independent and unimpaired); and the most commonly used Unified Parkinson's Disease Rating Scale (UPDRS). The UPDRS is a rating tool to follow the longitudinal course of Parkinson's Disease. It is made up of the 1) Mentation, Behavior, and Mood, 2) Activities of Daily Living and 3) Motor sections, with multiple parameters in each. Some sections require multiple grades assigned to each extremity. A total of 199 points are possible: 199 represents the worst (total) disability), 0—no disability.
A combination of genetic and environmental factors may cause or contribute to Parkinson's Disease. A rare hem of Parkinson's Disease is the result of an inherited genetic mutation, but the common form of Parkinson's Disease is sporadic. In some implementations, subjects who are the progeny of a Parkinson's Disease patient or of a carrier of the genetic mutation Parkinson's Disease, or who are identified as having Parkinson's Disease. (e.g., by a genetic test), are given a prophylactic dose of a TWEAK/TWEAK-R blocking agent, e.g., prior to showing physical symptoms of Parkinson's Disease.
Treatments for Parkinson's Disease include drugs such as levodopa and the dopamine agonists ropinirole, pramipexole, and pergolide can reduce muscle rigidity, improve speed and coordination of movement, and relieve tremor. Cell transplantation (e.g., embryonic stem cells and, adult stem cells) may also be a useful therapy for Parkinson's Disease. Any one or more of these treatments can be used in combination with a TWEAK/TWEAK-R blocking agent.
Symptoms of Parkinson's Disease usually appear after 80 percent or more of the dopamine-producing neurons in the substantia nigra have died, which significantly narrows the window of opportunity for preventive or protective treatments. The course of the disease varies widely. Some people have mild symptoms for many years, while others have severe symptoms and a quicker progression. A TWEAK/TWEAK-R blocking agent can be administered to subjects who show a depletion of dopamine producing neurons in the substantia nigra, e.g., a less than 80, 60, 40, or 30% depletion.
The efficacy and dosing of a TWEAK/TWEAK-R blocking agent can be evaluated in an animal model of Parkinson's Disease, e.g., treatment with 1-methyl-phenyl-1,2,3,6-tetrallydropyridine (MPTP). MPTP is a neurotoxin that damages nigrostriatal dopaminergic neurons in several mammalian species, including mice, and produces a Parkinsonian syndrome in humans and primates (U.S. Pat. No. 6,630,514). Another animal model of Parkinson's Disease is transgenic mice overexpressing human α-synucleire Neuronal expression of human α-synuelein results in progressive accumulation of α-syrtuelein and ubiquitin-immunoreactive inclusions in neurons in the neocortex, hippocampus, and substantia nigra. These alterations are associated with loss of dopaminergic terminals in the basal ganglia and with motor impairments. (Masliah et al. (2000) Science 287:1265-1269).
Huntington's Disease
A TWEAK/TWEAK-R blocking agent can be administered to treat Huntington's Disease (HD), which results from genetically-programmed degeneration of neurons in certain areas of the brain. HD is a genetic disease that is characterized by a generational expansion in the number of trinucleotide repeats in the gene encoding Huntingtin.
A TWEAK/TWEAK-R blocking agent can be used to delay onset, reduce severity, and reduce the rate of progression of Huntington's Disease. The agent can be used to treat long-term survivors as well as patients who exhibit more rapid disease progression. The agent can be administered to subjects that have a trinucleotide repeat length greater than 30, 35, 40, 45, or 50.
Some early symptoms of HD are mood swings; depression; irritability; or trouble driving, learning new things, remembering a fact, or making a decision. As the disease progresses, concentration on intellectual tasks becomes increasingly difficult and the patient may have difficulty feeding himself or herself and swallowing. A TWEAK/TWEAK-R blocking agent can be used to ameliorate one or more these symptoms, or to reduce the rate of disease progression. The blocking agent can also be administered at any stage, e.g., prior to the display of any symptoms, at a stage characterized by early symptoms, or at a later stage.
A genetic test, coupled with a complete medical history and neurological and laboratory tests, can be used to diagnose HD. Presymptomic testing is available for individuals who are at risk for carrying the mutated gene. In 1 to 3 percent of individuals with HD, no family history of HD can be found. In some implementations, subjects who are the progeny of Huntington disease patient or of a carrier of a Huntington's Disease mutation, or who are identified as having Huntington's Disease (e.g., by a genetic test), are given a prophylactic dose of a TWEAK/TWEAK-R blocking agent, e.g., prior to showing physical symptoms of Huntington's Disease.
A variety of methods are available to evaluate and/or monitor Huntington's Disease. A variety of clinical symptoms and indicia for the disease are known, Huntington's Disease causes a movement disorder, psychiatric difficulties, and cognitive changes. The degree, age of onset, and manifestation of these symptoms can vary. The movement disorder can include quick, random, dance-like movements called chorea.
One method for evaluating Huntington's Disease uses the Unified Huntington's Disease Rating Scale (UNDRS). It is also possible to use individual tests alone or in combination to evaluate if at least one symptom of Huntington's Disease is ameliorated. The UNDRS is described in Movement Disorders (vol. 11:136-142, 1996) and Marder et al. ((2000) Neurology 54:452-458). The UNDRS quantifies the severity of Huntington's Disease. It is divided into multiple subsections: motor, cognitive, behavioral, and functional. In one embodiment, a single subsection is used to evaluate a subject. These scores can be calculated by summing the various questions of each section. Some sections (such as chorea and dystonia) can include grading each extremity, face, bucco-oral-ligual, and trunk separately.
Exemplary motor evaluations include: ocular pursuit, saccade initiation, saccade velocity, dysarthria, tongue protrusion, finger tap ability, pronate/supinate, a fist-hand-palm sequence, rigidity of arms, bradykinesia, maximal dystonia (trunk, upper and lower extremities), maximal chorea (e.g., trunk, face, upper and lower extremities), gait, tandem walking, and retropulsion. An exemplary treatment can cause a change in the Total Motor Score 4 (TMS-4), a subscale of the UHDRS, e.g., over a one-year period.
Additional therapies include administering; bromocriptine, serotonin antagonists (e.g., tetrabenazin), tiapride (e.g., TIAPRIDAL®), or a NMDA receptor blocker. Any one or more of these treatments can be used in combination with a TWEAK/TWEAK-R blocking agent.
A number of animal model systems for Huntington's Disease are available. See, e.g., Brouillet (2000) Functional Neurology 15(0239-251; Ona et al. (1999) Nature 399:263-267; Bates et al, (1997) Hum. Mol. Genet, 6(10):1633-1637; Hausson et al. (2001) J. Neurochem, 78:694-703; and Rubinsztein (2002) Trends Genet, 18202-209 (a review on various animal and non-human models of HD).
Transgenic mice can be produced that express (in at least one cell) a human Huntingtin protein, a portion thereof, or fusion protein comprising human Huntingtin protein, or a portion thereof, with, for example, at least 36 giutamines (e.g., encoded by CAG repeats (alternatively, any number of the CAG repeats may be CAA) in the CAG repeat segment of exon 1 encoding the polyglutamine tract).
An example of such a transgenic mouse strain is the R6/2 line (Mangiarini et al. (1996) Cell 87:493-506). The R6/2 mice are transgenic Huntington's Disease mice, which over-express exon 1 of the human Huntingtin gene (under the control of the endogenous promoter). The exon 1 of the R6/2 human Huntingtin gene has expanded CAG/polyglutamine repeat lengths (150 CAG repeats on average). These mice develop a progressive, ultimately fatal, neurological disease with many features of human Huntington's Disease. Abnormal aggregates, constituted in part by the N-terminal part of Huntingtin (encoded by HD exon 1), are observed in R6/2 mice, both in the cytoplasm and nuclei of cells (Davies et al. (1997) Cell 90:537-548). For example, the human Huntingtin protein in the transgenic animal is encoded by a gene that includes at least 55 CAG repeats and more preferably about 150 CAG repeats.
The transgenic animals can develop a Huntington's Disease-like phenotype. These transgenic mice are characterized by reduced weight gain, reduced lifespan, and motor impairment characterized by abnormal gait, resting tremor, hindlimb clasping, and hyperactivity from 8 to 10 weeks after birth (for example the R6/2 strain; see Mangiarini at al. (1996) Cell 87:493-506), The phenotype worsens progressively toward hypokinesia. The brains of these transgenic mice also demonstrate neurochemical and histological abnormalities, such as changes in neurotransmitter receptors (glutamate, dopaminergic), decreased concentration of N-acetylaspartate marker of neuronal integrity), and reduced striatum and brain size. Accordingly, evaluating can include assessing parameters related to neurotransmitter levels, neurotransmitter receptor levels, brain, size, and striatum size. In addition, abnormal aggregates containing the transgenic part of or full-length human Huntingtin protein are present in the brain tissue of these animals (e.g., the R6/2 transgenic mouse strain). See, e.g., Mangiarini et al. (1996) Cell 87493-506; Davies et al. (1997) Cell 90:537-548; Brouillet (2000) Functional Neurol. 15(4):239-251; and Cha et al. (1998) Proc. Natl. Acad. Sci. USA 95:6480-6485.
Alzheimer's Disease
A TWEAK/TWEAK-R blocking agent can be administered to treat Alzheimer's Disease (AD), which is a progressive, neurodegenerative disease characterized in the brain by abnormal clumps (amyloid plaques) and tangled bundles of fibers (neurofibrillary tangles) composed of misplaced proteins. A TWEAK/TWEAK-R blocking agent can be used to delay onset, reduce severity, and reduce the rate of progression of Alzheimer's Disease. The agent can be used to treat long-term survivors as well as patients who exhibit more rapid disease progression. The severity and progression of, and improvement in, Alzheimer's Disease can be assessed by a number of criteria, including Activities of Daily Living scales, one of which is the Progressive Deterioration Scale (PDS), in which 29 questions are used to determine a patient's ability to perform functions such as interacting socially, travelling without getting lost, coping when the caregiver is away, and identifying or using common household objects such as telephones; the Mini-Mental State Examination (MMSE), which is a short collection of cognitive tests that examines several areas of coition and is easy to administer and score and is widely used to measure the onset, progression and severity of Alzheimer's Disease.
Age is the most important risk factor for AD; the number of people with the disease doubles every 5 years beyond age 65. Three genes have been discovered that cause early onset (familial) AD. Other genetic mutations that cause excessive accumulation of amyloid protein are associated with age-related (sporadic) AD. In some implementations, subjects who are the progeny of an AD patient or of a carrier of a mutation in a gene tied to AD, or who are identified as having Alzheimer's Disease (e.g., by a genetic test), are given a prophylactic dose of a TWEAK/TWEAK-R blocking agent, e.g., prior to showing physical symptoms of AD.
Symptoms of AD include memory loss, language deterioration, impaired ability to mentally manipulate visual information, poor judgment, confusion, restlessness, and mood swings. Eventually AD destroys cognition, personality, and the ability to function. The early symptoms of AD, which include forgetfulness and loss of concentration, are often missed because they resemble natural signs of aging. AD is a progressive disease, but its course can vary from 5 to 20 years. A TWEAK/TWEAK-R blocking agent can be used to ameliorate one or more these symptoms, or to reduce the rate of disease progression. Treatments for AD include tacrine (COGNEX®), donepezil (ARICEPT®), rivastigmine (EXELON®), and galantamine (REMINYL®), memantine (NAMENDATM), other drugs that may affect AD progression include nonsteroidal anti-inflammatory drugs (NSAIDS), statins, folic acid, gingko biloba, and vitamins E, B6, and B12. Any one or more of these treatments can be used in combination with a TWEAK/TWEAK-R blocking agent.
The efficacy and dosing of a TWEAK/TWEAK-R blocking agent can be evaluated in an animal model of AD, e.g., a transgenic mouse model of AD. For example, transgenic mice expressing a human or mouse APP or presenilin can be used. Some of these transgenic mice develop a progressive neurologic disorder generally is within a year from birth (see, e.g., U.S. Pat. Nos. 5,877,399; 6,037,521; 5,894,078; 5,850,003; and 5,898,094). Certain transgenic animal models have been described, for example, in U.S. Pat. Nos. 5,612,486; 5,387,742; 5,720,936; 5,877,015; and 5,811,633; and in Ganes et al. (1995) Nature 373:523. Preferred are animals that exhibit characteristics associated with the pathophysiology of AD. Administration of inhibitors to the transgenic mice described herein provides an alternative method for demonstrating the inhibitory activity of the compounds. Administration of the compounds in a pharmaceutically effective carrier and via an administrative route that reaches the target tissue in an appropriate therapeutic amount is also preferred.
Pharmaceutical Compositions
A TWEAK/TWEAK-R blocking agent (e.g., an antibody or a soluble TWEAK-R protein, e.g., TWEAK-R-Fc) can be formulated as a pharmaceutical composition, e.g., for administration to a subject to treat a neuronal disorder such as a mechanical neuronal trauma (e.g., SCI or TBI), a neurodegenerative disorder (e.g., ALS, Parkinson's Disease, Huntington's Disease, or Alzheimer's Disease), or other disorder described herein. Typically, a pharmaceutical composition includes a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The composition can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see, e.g., Berge, S. M. et al. (1977) J. Pharm. Sci. 66:1-19).
The TWEAK/TWEAK-R blocking agent can be formulated according to standard methods. Pharmaceutical formulation is a well-established art, and is further described, e.g., in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727); and Kibbe (ed.), Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3rd ed. (2000) (ISBN: 091733096X).
In one embodiment, the TWEAK/TWEAK-R blocking agent (e.g., an antibody or TWEAK-R-Fc) can be formulated with excipient materials, such as sodium chloride, sodium dibasic phosphate heptahydrate, sodium monobasic phosphate, and a stabilizer. It can be provided, for example, in a buffered solution at a suitable concentration and can be stored at 2-8° C.
The pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form can depend on the intended mode of administration and therapeutic application. Typically compositions for the agents described herein are in the form of injectable or infusible solutions.
Such compositions can be administered by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). The phrases “parenteral administration” and “administered parenterally” as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subeuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.
The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Sterile injectable solutions can be prepared by incorporating an agent described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating an agent described herein into a sterile vehicle that contains a 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 freeze drying that yields a powder of an agent described herein plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution 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 dispersion, and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
In certain embodiments, the TWEAK/TWEAK-R blocking agent may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
A TWEAK/TWEAK-R blocking agent (e.g., an antibody or a soluble TWEAK receptor protein) can be modified, e.g., with a moiety that improves its stabilization and/or retention in circulation, e.g., in blood, serum, or other tissues, e.g., by at least 1.5, 2, 5, 10, or 50-fold. The modified blocking agent can be evaluated to assess whether it can reach sites of neuronal damage, e.g., after a trauma or during the course of a neurological disorder (e.g., by using a labeled form of the blocking agent).
For example, the TWEAK/TWEAK-R blocking agent (e.g., an antibody or a soluble TWEAK receptor protein) can be associated with (e.g., conjugated to) a polymer, e.g., a substantially non-antigenic polymer, such as a polyalkylene oxide or a polyethylene oxide. Suitable polymers will vary substantially by weight. Polymers having molecular number average weights ranging from about 200 to about 35,000 Daltons (or about 1,000 to about 15,000, and 2,000 to about 12,500) can be used.
For example, a TWEAK or a TWEAK receptor binding antibody can be conjugated to a water-soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g., polyvinylalcohol or polyvinylpyrrolidone. A non-limiting list of such polymers includes polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained. Additional useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates; carbomers; and branched or unbranched polysaccharides.
When the TWEAK/TWEAK-R blocking agent (e.g., an antibody or a soluble TWEAK-R protein) is used in combination with a second agent, the two agents can be formulated separately or together. For example, the respective pharmaceutical compositions can be mixed, e.g., just prior to administration, and administered together or can be administered separately, e.g., at the same or different times.
Administration
The TWEAK/TWEAK-R blocking agent (e.g., an antibody or a soluble TWEAK-R protein) can be administered to a subject, e.g., a human subject, by a variety of methods. For many applications, the route of administration is one of: intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneally (IP), or intramuscular injection. In some cases, administration may be directly into the CNS, e.g., intrathecal or intracerebroventricular (ICV). The blocking agent can be administered as a fixed dose, or in a mg/kg dose.
The dose can also be chosen to reduce or avoid production of antibodies against the TWEAK/TWEAK-R blocking agent.
The route and/or mode of administration of the blocking agent can also be tailored for the individual case, e.g., by monitoring the subject, e.g., using tomographic imaging, neurological exam, and standard parameters associated with the particular disorder, e.g., criteria for assessing SCI, TBI, ALS, Parkinson's Disease, Huntington's Disease, or Alzheimer's Disease.
Dosage regimens are adjusted to provide the desired response, e.g., a therapeutic response or a combinatorial therapeutic effect. Generally, any combination of doses (either separate or co-formulated) of the TWEAK/TWEAK-R blocking agent (e.g., an antibody) (and optionally a second agent) can be used in order to provide a subject with the agent in bioavailable quantities. For example, doses in the range of 1 mg/kg-100 mg/kg, 0.5-20 mg/kg, or 1-10 mg/kg can be administered. Other doses can also be used.
Dosage unit form or “fixed dose” as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and optionally in association with the other agent, if used.
The TWEAK/TWEAK-R blocking agent may be administered at least once between about 10 minutes to about 48 hours, more preferably between about 10 minutes and 24 hours, more preferably within 3 hours, after the onset of symptoms or manifestation of a neuronal or other injury or pathology. For example, the agent may be administered to a patient suffering or at risk for a disorder described herein, e.g., SCI, TBI, ALS, Parkinson's Disease, Huntington's Disease, or Alzheimer's Disease. Single or multiple dosages may be given. Alternatively, or in addition, the blocking agent may be administered via continuous infusion. The treatment can continue for days, weeks, months or even years so as to minimize brain or spinal cord damage from the injury or disorder, to minimize damage from post-injury inflammatory events, and/or to prevent or minimize damage that might result from disease progression.
The TWEAK/TWEAK-R blocking agent can be administered, e.g., once or twice daily or about one to four times per week, e.g., for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The TWEAK/TWEAK-R blocking agent can be administered for months (e.g., 6, 12, 18, 24 or 36 months) or years (e.g., 1, 2, 3, 5, 10, or 15 years), e.g., to treat or prevent a degenerative disorder described herein. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, formulation, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of at subject with a therapeutically effective amount of a compound can include a single treatment or, preferably, can include a series of treatments. Animal models can also be used to determine a useful dose, e.g., an initial dose or a regimen.
If a subject is at risk for developing a neurodegenerative or other disorder described herein or an injury to the brain or spinal cord (e.g., SCI, TBI), the blocking agent can be administered before the onset of an injury or disorder as a preventative measure. The duration of such preventative treatment can be a single dosage of the blocking agent or the treatment may continue (e.g., multiple dosages). For example, a subject at risk for the disorder or who has a predisposition for the disorder may be treated with the blocking agent for days, weeks, months, or even years so as to prevent the injury or disorder from occurring.
Surgery in or near the brain or spinal cord can put a subject at risk for SCI or TBI. The blocking agent can be administered prior to, during, and after such surgery in or near the brain or spinal cord, e.g., to minimize neuronal damage caused by the surgery as well as pre- or post-operative events.
A pharmaceutical composition may include a “therapeutically effective amount” of an agent described herein. Such effective amounts can be determined based on the effect of the administered agent, or the combinatorial effect of agents if more than one agent is used. A therapeutically effective amount of an agent may also vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual, e.g., amelioration of at least one disorder parameter or amelioration of at least one symptom of the disorder. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.
Devices and Kits
Pharmaceutical compositions that include the TWEAK/TWEAK-R blocking agent (e.g., an antibody or soluble TWEAK-R) can be administered with a medical device. The device can designed with features such as portability, room temperature storage, and ease of use so that it can be used in emergency situations, e.g., by an untrained subject or by emergency personnel in the field, removed to medical facilities and other medical equipment. The device can include, e.g., one or more housings for storing pharmaceutical preparations that include TWEAK/TWEAK-R blocking agent, and can be configured to deliver one or more unit doses of the blocking agent.
For example, the pharmaceutical composition can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. Many other devices, implants, delivery systems, and modules are also known.
A TWEAK/TWEAK-R blocking agent (e.g., an antibody or soluble TWEAK-R protein) can be provided in a kit. In one embodiment, the kit includes (a) a container that contains a composition that includes a TWEAK or a TWEAK receptor blocking agent, and optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the agents for therapeutic benefit. In an embodiment, the kit also includes a second agent for treating a neuronal disorder such as a mechanical neuronal trauma (e.g., SCI or TBI), a neurodegenerative disorder (e.g., ALS, Parkinson's Disease, Huntington's Disease, or Alzheimer's Disease), or other disorder described herein. For example, the kit includes a first container that contains a composition that includes the TWEAK/TWEAK-R blocking agent, and a second container that includes the second agent.
The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to methods of administering the TWEAK/TWEAK-R blocking agent (e.g., an antibody or a soluble TWEAK-R protein), e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein), to treat a subject who has had or who is at risk for a neuronal disorder such as a mechanical neuronal trauma (e.g., SCI or TBI), a neurodegenerative disorder (e.g., ALS, Parkinson's Disease, Huntington's Disease, or Alzheimer's Disease), or other disorder described herein. The information can be provided in a variety of formats, including printed text, computer readable material, video recording, audio recording, or information that provides a link or address to substantive material located on the world wide web.
In addition to the blocking agent, the composition in the kit can include other ingredients, such as a solvent or buffer, a stabilizer, or a preservative. The blocking agent can be provided in any forme e.g., liquid, dried, or lyophilized form, preferably substantially pure and/or sterile. When the agent is provided in a liquid solution, the liquid solution preferably is an aqueous solution. When the agent is provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit.
The kit can include one or more containers for the composition or compositions containing the agent. In some embodiments, the kit contains separate containers, dividers, or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial, or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the agent. The containers can include a combination unit dosage, e.g., a unit that includes both the TWEAK or a TWEAK receptor blocking agent and the second agent, e.g., in a desired ratio. For example, the kit includes a plurality of syringes, ampules, foil packets, blister packs, or medical devices, e.g., each containing a single combination unit dose. The containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.
The kit optionally includes a device suitable for administration of the agent (e.g., in a pharmaceutical composition), e.g., a syringe or other suitable delivery device. The device can be provided pre-loaded with one or a combination of agents or can be empty, but suitable for loading.
Nucleic Acid and Protein Analysis
Numerous methods for detecting TWEAK or a TWEAK-R protein and nucleic acid are available to the skilled artisan, including antibody-based methods for protein detection (e.g., Western blot or ELISA), and hybridization-based methods for nucleic acid detection (e.g., PCR or Northern blot).
Arrays are particularly useful molecular tools for characterizing a sample, e.g., a sample from a subject. For example, an array having capture probes for multiple genes, including probes for TWEAK and a TWEAK receptor, or for multiple proteins, can be used in a method described herein. Altered expression of TWEAK or TWEAK receptor nucleic acids and/or protein can be used to evaluate a sample, e.g., a sample from a subject, e.g., to evaluate, a disorder described herein.
Arrays can have many addresses, e.g., locatable sites, on a substrate. The featured arrays can be configured in a variety of formats, non-limiting examples of which are described below. The substrate can be opaque, translucent, or transparent. The addresses can be distributed, on the substrate in one dimension, e.g., a linear array; in two dimensions, e.g., a planar array; or in three dimensions, e.g., a three-dimensional array. The solid substrate may be of any convenient shape or form, e.g., square, rectangular, ovoid, or circular.
Arrays can be fabricated by a variety of methods, e.g., photolithographic methods (see, e.g., U.S. Pat. Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Pat. No. 5,384,261), pin-based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead-based techniques (e.g., as described in PCT App, US/93/04145).
The capture probe can be a single-stranded nucleic acid, a double-stranded nucleic acid (e.g., which is denatured prior to or during hybridization), or a nucleic acid having a single-stranded region and a double-stranded region. Preferably, the capture probe is single-stranded. The capture probe can be selected by a variety of criteria, and preferably is designed by a computer program with optimization parameters. The capture probe can be selected to hybridize to a sequence rich (e.g., non-homopolymeric) region of the gene. The Tm of the capture probe can be optimized by prudent selection of the complementarity region and length. Ideally, the Tm of all capture probes on the array is similar, e.g., within 20, 10, 5, 3, or 2° C. of one another.
The isolated nucleic acid is preferably mRNA that can be isolated by routine methods, e.g., including DNase treatment to remove genomic DNA and hybridization to an oligo-dT coupled solid substrate (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y). The substrate is washed and the mRNA is eluted.
The isolated mRNA can be reversed transcribed and optionally amplified, e.g., by rtPCR, e.g., as described in U.S. Pat. No. 4,683,202. The nucleic acid can be an amplification product, e.g., from PCR (U.S. Pat. Nos. 4,683,196 and 4,683,202); rolling circle amplification (“RCA,” U.S. Pat. No. 5,714,320), isothermal RNA amplification or NASBA (U.S. Pat. Nos. 5,130,238; 5,409,818; and 5,554,517), and strand displacement amplification (U.S. Pat. No. 5,455,166). The nucleic acid can be labeled during amplification, by the incorporation of a labeled nucleotide. Examples of preferred labels include fluorescent labels, e.g., red-fluorescent dye Cy5 (Amersham) or green-fluorescent dye Cy3 (Amersham), and chemiluminescent labels, e.g., as described in U.S. Pat. No. 4,277,437. Alternatively, the nucleic acid can be labeled with biotin, and detected after hybridization with labeled streptavidin, e.g., streptavidin-phycoerythrin (Molecular Probes).
The labeled nucleic acid can be contacted to the array. In addition, a control nucleic acid or a reference nucleic acid can be contacted to the same array. The control nucleic acid or reference nucleic acid can be labeled with a label other than the sample nucleic acid, one with a different emission maximum. Labeled nucleic acids can be contacted to an array under hybridization conditions. The array can be washed and then imaged to detect fluorescence at each address of the array.
The expression level of a TWEAK or TWEAK-R protein can be determined using an antibody specific for the polypeptide (e.g., using a Western blot or an ELISA assay). Moreover, the expression levels of multiple proteins, including TWEAK and a TWEAK receptor, can be rapidly determined in parallel using a polypeptide array having antibody capture probes for each of the polypeptides. Antibodies specific for a polypeptide can be generated by a method described herein (see “Antibody Generation”). The expression level of a TWEAK or TWEAK receptor can be measured in a subject (e.g., in vivo imaging) or in a biological sample from a subject (e.g., blood, serum, plasma, or cerebral spinal fluid).
A low-density (96-well format) protein array has been developed in which proteins are spotted onto a nitrocellulose membrane (Ge (2000) Nucleic. Acids Res. 28, e3, I-VII). A high-density protein array (100,000 samples within 222×222 mm) used for antibody screening was formed by spotting proteins onto polyvinylidene difluoride (PVDF) (Lueking et al. (1999) Anal. Biochem, 270:103-111), See also, e.g., Mendoza et al. (1999) Biotechniques 27:778-788; MacBeath and Schreiber (2000) Science 289:1760-1763; and De Wildt et al. (2000) Nat. Biotech, 18:989-994. These art-known methods and others can be used to generate an array of antibodies for detecting the abundance of polypeptides in a sample. The sample can be labeled, e.g., biotinylated, for subsequent detection with streptavidin coupled to a fluorescent label. The array can then be scanned to measure binding at each address.
The nucleic acid and polypeptide arrays of the invention can be used in a wide variety of applications. For example, the arrays can be used to analyze a patient sample. The sample is compared to data obtained previously, e.g., known clinical specimens or other patient samples. Further, the arrays can be used to characterize a cell culture sample, e.g., to determine a cellular state after varying a parameter, e.g., exposing the cell culture to an antigen, a transgene, or a test compound.
The expression data can be stored in a database, e.g., a relational database such as a SQL database (e.g., Oracle or Sybase database environments). The database can have multiple tables. For example, raw expression data can be stored in one table, wherein each column corresponds to a gene being assayed, e.g., an address or an array, and each row corresponds to a sample. A separate table can store identifiers and sample information, e.g., the batch number of the array used, date, and other quality control information.
Expression profiles obtained from gene expression analysis on an array can be used to compare samples and/or cells in a variety of states as described in Golub et al. ((1999) Science 286:531). In one embodiment, expression (e.g., mRNA expression or protein expression) information for a gene encoding TWEAK and/or a gene encoding a TWEAK receptor are evaluated, e.g., by comparison to a value, e.g., a reference value. Reference values can be obtained from a control, e.g., a reference subject. Reference values can also be obtained from statistical analysis, e.g., to provide a reference value for a cohort of subjects, e.g., age and gender matched subjects, e.g., normal subjects or subjects who have sustained a neuronal or other injury or pathology in the brain or spinal cord or who have a disorder described herein. Statistical similarity to a particular reference (e.g., to a reference for a risk-associated cohort) or a normal cohort can be used to provide an assessment (e.g., an indication of risk of neuronal injury or a neurological disorder) to a subject, e.g., a subject who has not sustained a prior neuronal or other injury to the brain or spinal cord, a subject who has not been diagnosed with a disorder described herein, a subject who has a risk for a neuronal disorder such as a neurodegenerative disorder (e.g., ALS, Parkinson's Disease, Huntington's Disease, or Alzheimer's Disease), or other disorder described herein (e.g., a genetic predisposition) or a risk for a disorder described herein, or a subject who has sustained a neuronal or other injury to the brain or spinal cord.
Subjects suitable for treatment can also be evaluated for expression and/or activity of TWEAK and/or a TWEAK receptor. Subjects can be identified as suitable for treatment if the expression and/or activity for TWEAK and/or a TWEAK receptor is elevated relative to a reference, e.g., reference value, e.g., a reference value associated with normal.
Subjects who are being administered an agent described herein or other treatment for a neuronal disorder such as a mechanical neuronal trauma (e.g., SCI or TBI), a neurodegenerative disorder (e.g., ALS, Parkinson's Disease, Huntington's Disease, or Alzheimer's Disease), or other disorder described herein, can be evaluated as described for expression and/or activity of TWEAK and/or a TWEAK receptor. The subject can be evaluated at multiple times, e.g., at multiple times during a course of therapy, e.g., during a therapeutic regimen. Treatment of the subject can be modified depending on how the subject is responding to the therapy. For example, a reduction in TWEAK and/or TWEAK receptor expression or activity can be indicative of responsiveness.
Particular effects mediated by an agent may show a difference (e.g., relative to an untreated subject, control subject, or other reference) that is statistically significant (e.g., P value<0.05 or 0.02). Statistical significance can be determined by any art known method. Exemplary statistical tests include: the Students T-test, Mann Whitney U non-parametric test, and Wilcoxon non-parametric statistical test. Some statistically significant relationships have a P value of less than 0.05 or 0.02.
In Vivo Imaging
TWEAK and/or TWEAK-R blocking agents (e.g., antibodies) provide a method for detecting the presence of TWEAK and/or a TWEAK receptor (e.g., Fn14) in vivo (e.g., in viva imaging in a subject), respectively. The method can be used to evaluate (e.g., diagnose, localize, or stage) a condition or a neuronal disorder such as a mechanical neuronal trauma (e.g., SCI or TRI), a neurodegenerative disorder (e.g., ALS, Parkinson's Disease, Huntington's Disease, or Alzheimer's Disease), or other disorder described herein. The method includes: (i) administering to a subject (and optionally a control subject) a TWEAK or TWEAK receptor binding agent (e.g., a blocking agent that binds to TWEAK or a TWEAK receptor, e.g., an antibody or antigen binding fragment thereof, although such agents need not be blocking agents), under conditions that allow interaction of the binding agent and TWEAK or a TWEAK receptor to occur; and (ii) detecting localization of the binding agent in the subject. The method can be used to detect the location of TWEAK or TWEAK receptor expressing cells. A statistically-significant increase in the amount of the complex in the subject relative to the reference, e.g., the control subject or subject's baseline, can be a factor that may lead to a diagnosis of neuronal or other injury or pathology in the brain or spinal cord or of a neurological disorder or risk for such an injury or disorder.
Preferably, the TWEAK and for TWEAK receptor binding agent used in the in vivo (and also in vitro) diagnostic methods is directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound binding agent. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. In one embodiment, the TWEAK or TWEAK-R binding protein is coupled to a radioactive ion, e.g., indium (111In), iodine (131I or 125I), yttrium (90Y), actinium (225Ac), bismuth (212Bi or 213Bi), sulfur (35S), carbon (14C), tritium (3H), rhodium (188Rh), or phosphorous (32P). In another embodiment, the TWEAK/TWEAK-R binding protein is labeled with an NMR contrast agent.
In one aspect, the invention features a method of imaging vasculature in a patient who is at risk for neuronal or other injury or pathology in the brain or spinal cord or for developing a neurological disorder (e.g., a neurodegenerative disorder), has experienced such an injury, and/or has a neuronal or other disorder of the brain or spinal cord. The method includes: providing an agent that binds to TWEAK or a TWEAK receptor, e.g., an agent described herein, wherein the protein is physically associated to an imaging agent; administering the agent to a patient, e.g., with a risk for neuronal or other injury to the brain or spinal cord; or for a neurological disorder and imaging the patient, e.g., to detect TWEAK or TWEAK receptor expressing cells.
Methods of Evaluating Genetic Material
There are numerous methods for evaluating genetic material to provide genetic information. These methods can be used to evaluate a genetic locus that includes a gene encoding TWEAK or a gene encoding a TWEAK receptor, as well as other loci. The methods can be used to evaluate one or more nucleotides, e.g., a coding or non-coding region of the gene, e.g., in a regulatory region (e.g., a promoter, a region encoding an untranslated region or intron, and so forth).
Nucleic acid samples can be analyzed using biophysical techniques (e.g., hybridization, electrophoresis, and so forth), sequencing, enzyme-based techniques, and combinations thereof. For example, hybridization of sample nucleic acids to nucleic acid microarrays can be used to evaluate sequences in an mRNA population and to evaluate genetic polymorphisms. Other hybridization-based techniques include sequence specific primer binding (e.g., PCR or LCR); Southern analysis of DNA, e.g., genomic DNA; Northern analysis of RNA, e.g., mRNA; fluorescent probe-based techniques (sec, e.g., Beaudet et al. (2001) Genome Res. 11(4):600-608); and allele specific amplification. Enzymatic techniques include, restriction enzyme digestion; sequencing; and single base extension (SBE). These and other techniques are well known to those skilled in the art.
Electrophoretic techniques include capillary electrophoresis and Single-Strand Conformation Polymorphism (SSCP) detection (see, e.g., Myers et al. (1985) Nature 313:495-498 and Ganguly (2002) Hum Mutat. 19(4)334-342). Other biophysical methods include denaturing high pressure liquid chromatography (DHPLC).
In one embodiment, allele specific amplification technology that depends on selective PCR amplification may be used to obtain genetic information. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al (1989) Nucl. Acids Res. 17:2437-2448) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce, polymerase extension (Prosser (1993) Trends Biotechnol. 11:238-246). In addition, it is possible to introduce a restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al., (1992) Mol. Cell Probes 6:1). In another embodiment, amplification can be performed using Taq ligase for amplification (Barmy (1991) Proc. Natl. Acad. Sci. USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3′ end of the 5′ sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
Enzymatic methods for detecting sequences include amplification-based methods such as the polymerase chain reaction (PCR; Saiki et al. (1985) Science 230:1350-1354) and ligase chain reaction (LCR; Wu et al. (1989) Genomics 4:560-569; Barringer et al. (1990) Gene 1989:117-122; F. Barany (1991) Proc. Natl. Acad. Sci. USA 1988:189-193); transcription-based methods utilizing RNA synthesis by RNA polymerases to amplify nucleic acid (U.S. Pat. Nos. 6,066,457; 6,132,997; and 5,716,785; Sarkar et al. (1989) Science 244:331-334; Stoller et al. (1988) Science 239:491); NASBA (U.S. Pat. Nos. 5,130,238; 5,409,818; and 5,554,517); rolling circle amplification (RCA; U.S. Pat. Nos. 5,854,033 and 6,143,495), and strand displacement amplification (SDA; U.S. Pat. Nos. 5,455,166 and 5,624,825). Amplification methods can be used in combination with other techniques.
Other enzymatic techniques include sequencing using polymerases, e.g., DNA polymerases and variations thereof, such as single base extension technology. See, e.g., U.S. Pat. Nos. 6,294,336; 6,013,431; and 5,952,174.
Fluorescence-based detection can also be used to detect nucleic acid polymorphisms. For example, different terminator ddNTPs can be labeled with different fluorescent dyes. A primer can be annealed near or immediately adjacent to a polymorphism, and the nucleotide at the polymorphic site can be detected by the type (e.g., “color”) of the fluorescent dye that is incorporated.
Hybridization to microarrays can also be used to detect polymorphisms, including SNPs. For example, a set of different oligonucleotides, with the polymorphic nucleotide at varying positions within the oligonucleotides, can be positioned on a nucleic acid array. The extent of hybridization as a function of position and hybridization to oligonucleotides specific for the other allele can be used to determine whether a particular polymorphism is present. See, e.g., U.S. Pat. No. 6,066,454.
In one implementation, hybridization probes can include one or more additional mismatches to destabilize duplex formation and sensitize the assay. The mismatch may be directly adjacent to the query position, or within 10, 7, 5, 4, 3, or 2 nucleotides of the query position. Hybridization probes can also be selected to have a particular Tm e.g., between 45-60° C., 55-65° C., or 60-75° C., In a multiplex assay, Tm's can be selected to be within 5, 3, or 2° C. of each other.
It is also possible to directly sequence the nucleic acid for a particular genetic locus, e.g., by amplification and sequencing, or amplification, cloning and sequencing. High throughput automated (e.g., capillary or microchip based) sequencing apparati can be used. In still other embodiments, the sequence of a protein of interest is analyzed to infer its genetic sequence. Methods of analyzing a protein sequence include protein sequencing, mass spectroscopy, sequence/epitope specific immunoglobulins, and protease digestion.
Any combination of the above methods can also be used. The above methods can be used to evaluate any genetic locus, e.g., in a method for analyzing genetic information from particular groups of individuals or in a method for analyzing a polymorphism associated with a neuronal or other injury or pathology in the brain or spinal cord or other neurological disorder, e.g., in a gene encoding TWEAK or a TWEAK receptor.
EXAMPLES
Example 1
TWEAK and Fn14 Levels in FALS
FIG. 1 shows that Fn14 is upregulated in an animal model of ALS, indicating that blocking the TWEAK/TWEAK-R pathway can be used as a therapeutic strategy to treat ALS.
Example 2
MPTP Model for Parkison's Disease
The MPTP mouse model is widely-used animal model for Parkinson's Disease. It is an excitotoxic model of dopaminergic neurodegeneration. MPTP is injected IP and MPTP intoxication causes death of dopaminergic neurons in substantia nigra (SN). Gliosis is measured on day 1, 2 and tyrosine hydroxylase (TH)+ cell survival in substantia nigra on day 7. As shown in FIG. 2, Fn14 knock-out mice are protected against MPTP toxicity.
The effect of Fn14 deficiency on microglial activation in the MPTP model was also analyzed. Mac-1 and PCNA double positive cells in the substantia nigra were counted on day 1 and day 2 after MPTP injection. As shown in FIG. 3, Fn14 deficiency reduces microglial activation after MPTP injection.
The data indicate that blocking the TWEAK/TWEAK receptor pathway can be used as a therapeutic strategy to treat Parkinson's disease.
Example 3
An Exemplary Sequence of a Human Tweak Protein is as Follows









(SEQ ID NO: 1)



MAARRSQRRR GRRGEPGTAL LVPLALGLGL ALACLGLLLA



 



VVSLGSRASL SAQEPAQEEL VAEEDQDPSE LNPQTEESQD



 



PAPFLNRLVR PRRSAPKGRK TRARRAIAAH YEVHPRPGQD



 



GAQAGVLGTV SGWEEARINS SSPLRYNRQI GEFIVTRAGL



 



YYLYCQVHFD EGKAVYLKLD LLVDGVLALR CLEEFSATAA



 



SSLGPQLRLC QVSGLLALRP GSSLRIRTLP WAHLKAAPFL



 



TYFGLFQVH






An exemplary sequence of a human Fn14 protein is as follows:









(SEQ ID NO: 2)



MARGSLRRLL RLLVIALWLA LLRSVAGEQA PGTAPCSRGS



 



SWSADLDKCM DCASCPARPH SDFCLGCAAA PPAPFRLLWP



 



ILGGALSLTF VLGLLSGFLV WRRCRRREKF TTPIEETGGE



 



GCPAVALIQ






A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

## Claims

1. A method of treating a human subject who has Parkinson's Disease, comprising administering to the human subject a therapeutically effective amount of an anti-TWEAK (TNF-like weak inducer of apoptosis) antibody or antigen-binding fragment thereof that blocks TWEAK / TWEAK-R (TNF-like weak inducer of apoptosis-receptor) interaction.

2. The method of claim 1, wherein the anti-TWEAK antibody or antigen-binding fragment thereof is humanized.

3. The method of claim 1, wherein the anti-TWEAK antibody or antigen-binding fragment thereof is monoclonal.

4. The method of claim 1, wherein a full-length anti-TWEAK antibody is administered.

5. The method of claim 4, wherein the full-length anti-TWEAK antibody is a full-length IgG antibody.

6. The method of claim 1, wherein the anti-TWEAK antibody or antigen-binding fragment thereof is human.

7. The method of claim 1, wherein the treatment comprises administering the anti-TWEAK antibody or antigen-binding fragment thereof in combination with a second agent for treating Parkinson's disease.

8. The method of claim 7, wherein the second agent is a dopamine agonist or an MAO-B inhibitor.

9. The method of claim 7, wherein the second agent is selected from the group consisting of levodopa, ropinrole, pramipexole, pergolide, and R(+)-N-propargyl-1-aminoindan.

10. The method of claim 1, wherein the TWEAK-R is Fn14.

11. The method of claim 10, wherein the anti-TWEAK antibody or antigen-binding fragment thereof is administered via a route selected from the group consisting of: intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal injection or infusion, and directly into the central nervous system.

12. The method of claim 11, wherein the anti-TWEAK antibody or antigen-binding fragment thereof is administered via a subcutaneous route.

13. The method of claim 11, wherein the anti-TWEAK antibody or antigen-binding fragment thereof is administered via an intravenous route.

14. The method of claim 10, wherein the anti-TWEAK antibody or antigen-binding fragment thereof is in a delivery formulation selected from the group consisting of a tablet, pill, powder, dispersion, suspension, liposome, suppository, liquid, emulsion, implant, and sustained-release formulation.

15. The method of claim 14, wherein the anti-TWEAK antibody or antigen-binding fragment thereof is in a liquid delivery formulation.

16. The method of claim 10, wherein the anti-TWEAK antibody or antigen-binding fragment thereof is humanized.

17. The method of claim 10, wherein the anti-TWEAK antibody or antigen-binding fragment thereof is human.

18. The method of claim 10, wherein a full-length anti-TWEAK IgG antibody is administered.

19. The method of claim 10, wherein the treatment comprises administering the anti-TWEAK antibody or antigen-binding fragment thereof in combination with a second agent for treating Parkinson's disease.

20. The method of claim 19, wherein the second agent is a dopamine agonist or an MAO-B inhibitor.

21. The method of claim 19, wherein the second agent is selected from the group consisting of levodopa, ropinrole, pramipexole, pergolide, and R(+)-N-propargyl-1-aminoindan.

22. The method of claim 10, wherein an antigen-binding fragment of an anti-TWEAK antibody is administered.

23. The method of claim 22, wherein the antigen-binding fragment of the anti-TWEAK antibody is fused to an antibody Fc region.

24. The method of claim 22, wherein the antigen-binding fragment of the anti-TWEAK antibody comprises a single chain antibody, a Fab fragment, a F(ab′)2 fragment, a Fd fragment, a Fv fragment, or a dAb fragment.