METHODS AND COMPOSITIONS FOR NERVE REGENERATION
Methods and compositions for modulating growth of a neuron with a Wnt, a Wnt-like substance, and/or a chemical compound affecting a Wnt signaling pathway are disclosed. Also disclosed are methods for identifying a substance that modulates growth of a neuron by obtaining a candidate substance and contacting the candidate substance with the neuron are disclosed and methods for modulating growth of a neuron in a subject using a Wnt, a Wnt-like substance, and/or a chemical compound affecting a Wnt signaling pathway. The Wnt, Wnt-like substance, and/or chemical compounds affecting a Wnt signaling pathway can be delivered to the subject using gene therapy techniques. Also disclosed are pharmaceutical compositions for modulating growth of a neuron in a mammal that include a Wnt or a Wnt-like substance. Methods and compositions for inhibiting growth of a neuron are also disclosed.
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This application is a divisional of U.S. application Ser. No. 10/847,972 filed May 17, 2004, which claims the benefit of U.S. Provisional Application No. 60/470,913 filed May 15, 2003, and both are incorporated herein by reference in their entireties.
BACKGROUND OF THE INVENTION1. Field of the Invention
The present invention relates generally to the fields of molecular biology, cell biology, pharmacology, developmental neuroscience, neurology, neurosurgery and regenerative biology. More particularly, it concerns methods and compositions for modulating regeneration of a nerve cell using a Wnt, a Wnt-like substance, and/or a chemical compound affecting a Wnt signaling pathway. It also concerns methods and compositions for inhibiting growth of a neuron using inhibitors of neuronal growth that act via the Wnt signaling pathways, such as a Secreted Frizzled-Related Protein (sFRP), sFRP-like substance, Ryk, or Ryk-like substance.
2. Description of Related Art
The central nervous system (CNS) is connected by ascending sensory pathways and descending motor or regulatory pathways. In the CNS, somatosensory pathways ascend to the brain centers, and motor pathways controlling body movement descend from the brain to the spinal cord (Fitzgerald, 1996). The molecular mechanisms of axonal connections along the longitudinal axis of the CNS have remained a long-standing mystery.
Unlike the peripheral nervous system, damage to the central nervous system axons, such as spinal cord axons cannot be repaired, causing permanent impairment of neural function, such as in paralysis. The spinal cord serves important functions in the central nervous system. One such function is to allow communication of the body and the brain. The nerve fibers within the spinal cord carry messages to and from the brain to other parts of the body. In general sensory information from the body travels along the spinal cord up to the brain and instruction from the brain, such as motor command, travels along the spinal cord down from the brain. Thus, the spinal cord can be compared to a telephone cable, which connects the central office (brain) to the individual homes.
The term spinal cord injury refers to any injury of the neurons within the spinal canal. Spinal cord injury can occur from either trauma or disease to the vertebral column or the spinal cord itself. Most spinal cord injuries are the result of trauma to the vertebral column causing a fracture of the bone, or tearing of the ligaments with displacement of the bony column producing a pinching of the spinal cord. The majority of broken necks and broken backs, or vertebral fractures, do not cause any spinal cord damage; however, in 10-14% of the cases where a vertebral trauma has occurred, the damage is of such severity it results in damage to the spinal cord.
Spinal cord injury primarily occurs in young men with the greatest number of injuries occurring in the 16-30 age group. Patients with a spinal cord injury are designated as having tetraplegia (preferred to quadriplegia) or paraplegia. Tetraplegia refers to injuries to the cervical spinal cord and paraplegia refers to injuries below the cervical spinal cord. Patients with tetraplegia are slightly more common (51.7%) than patients with paraplegia. The majority of spinal cord injuries, about 37.4%, are sustained during a motor vehicle accident. Acts of violence are the second most common cause at 25.9%, falls are third at 21.5% and sports injuries are fourth at 7.1%.
It is estimated that the annual incidence of spinal cord injury (SCI), not including those who die at the scene of the accident, is approximately 40 cases per million population in the U.S., or approximately 11,000 new cases each year. The number of people in the U.S. who are alive today and who have SCI has been estimated to be between 721 and 906 per million population. This corresponds to between 183,000 and 230,000 persons.
Treatment options for patients with spinal cord injuries are limited. Often, patients with SCI are left with severe, permanent disabilities. A major reason for the limited availability of treatment options is the fact that there is little known about factors that can control and modulate nerve growth and regeneration following spinal cord injury. For example, the precise molecular mechanisms that guide axons along the anterior-posterior (A-P) axis of the spinal cord are unknown.
Axonal connections are patterned along the A-P and dorsal-ventral (D-V) neuraxes, wiring a large number of neurons into an intricate network. Axon guidance along the D-V axis has been a major focus of study in a number of experimental systems in recent years (Tessier-Lavigne and Goodman, 1996; Dickson, 2002). Much work has concentrated on the question of how axons are guided towards and away from the ventral midline and how midline crossing is regulated. Guidance molecules, such as Netrin-1 and members of the Slit and Semaphorin families, play pivotal roles in the dorsal-ventral guidance of axons (Tessier-Lavigne and Goodman, 1996; Dickson, 2002). The nature of the anterior-posterior guidance cues remains an enigma. Four classes of axon guidance molecules have been described (Tessier-Lavigne and Goodman, 1996): long-range attractants, long-range repellents, contact-mediated attractants and contact-mediated repellents. It is currently unknown whether a general gradient of attractant(s) or repellent(s) along the anterior-posterior axis guides axons to grow along this axis, or whether this guidance is mediated by more regional or segmental cues. The question of axon guidance along the A-P axis is of particularly interest in the spinal cord, where multiple classes of axons project either anteriorly or posteriorly along the length of the spinal cord. For example, somatosensory pathways ascend from the spinal cord to the brain and motor pathways descend from the brain to the spinal cord, with both the ascending and descending pathways carrying topographic information (FitzGerald, 1996).
The dorsal spinal cord commissural neurons form several ascending somatosensory pathways, such as the spinothalamic tracts, which send pain and temperature sensations to the brain (Ramon y Cajal, 1893; Altman and Bayer, 1984). The cell bodies of commissural neurons are located in the dorsal spinal cord. During embryonic development, commissural neurons project axons to the ventral midline. Once they reach the floor plate, they cross the midline and enter the contralateral side of the spinal cord. After midline crossing, commissural axons make a remarkably sharp anterior turn towards the brain (Ramon y Cajal, 1893; Altman and Bayer 1984; Tessier-Lavigne, 1994). All dorsal spinal cord commissural axons along the entire anterior-posterior length of the spinal cord project anteriorly after midline crossing. The initial ventral growth of the commissural axons is controlled by a gradient of a diffusible chemoattractant, Netrin-1 (Serafini et al., 1994; Kennedy et al., 1994; Serafini et al., 1996). As the axons cross the midline, they lose responsiveness to Netrin-1 (Shirasaki et al., 1998). Interestingly, while losing responsiveness to Netrin-1 during midline crossing, commissural axons gain responsiveness to several chemorepellents, which are located in the midline and the ventral spinal cord (Zou et al., 2000). These repellents help to expel the axons from the midline and to turn axons from their dorsal-ventral trajectory into their longitudinal pathways along the anterior-posterior axis by preventing axons from overshooting into the contralateral ventral spinal cord and recrossing the floor plate; the axons thus become “squeezed” into their longitudinal pathway (Zou et al., 2000). The expression pattern of the Slits and Semaphorins identified in these studies have been examined, but no anterior-posterior gradient of these chemorepellents in the spinal cord has been identified, suggesting that these repellents do not control anterior-posterior pathfinding.
Wnt polypeptides are secreted cysteine-rich glycosylated polypeptides that play a role in the development of a wide range of organisms. The Wnt family of polypeptides bind to an extracellular domain of a family of cell surface proteins called Frizzled receptors, and may play a role in embryonic induction, generation of cell polarity, and specification of cell fate.
Wnts are encoded by a large gene family, whose members have been found in round worms, insects, cartilaginous fish and vertebrates (Sidow, 1994). Wnts are thought to function in a variety of developmental and physiological processes since many diverse species have multiple conserved Wnt genes (McMahon, 1992; Nusse and Varmus, 1992). The Wnt growth factor family includes at least 18 genes identified in the human by cDNA cloning (see, e.g., Vant Veer et al., 1984; Miller, 2001).
Wnts may play a role in local cell signaling and neurogenesis. Biochemical studies have shown that much of the secreted Wnt protein can be found associated with the cell surface or extracellular matrix rather than freely diffusible in the medium (Papkoff and Schryver, 1990; Bradley and Brown, 1990). Studies of mutations in Wnt genes have indicated a role for Wnts in growth control and tissue patterning. In Drosophila, wingless (wg) encodes a Wnt gene (Rijsenijk et al., 1987) and wg mutations alter the pattern of embryonic ectoderm, neurogenesis, and imaginal disc outgrowth (Morata and Lawrence, 1977; Baker, 1988; Klingensmith and Nusse, 1994). Knock-out mutations in mice have shown Wnts to be essential for brain development (McMahon and Bradley, 1990; Thomas and Cappechi, 1990). However, a role for Wnts in mammalian directional axonal growth regulation in the spinal cord has not previously been suggested or considered.
The identification of modulators of neuronal growth and regeneration following SCI could be applied in new forms of treatment of patients with this debilitating condition. The identification of modulators of neuronal growth and regeneration could also be applied in the treatment of patients with other disorders involving neuronal dysfunction, such as neurodegenerative diseases. Agents that can promote axonal growth along the A-P axis following injury to the spinal cord may be applied to help prevent the permanent paralysis that is often associated with SCI. Therefore, there is a need for better treatments of SCI, and a greater understanding of modulators of neuronal growth and regeneration might lead to improved methods of treatment of this devastating disorder.
SUMMARY OF THE INVENTIONThe inventor has found that Wnt proteins play a general role in anterior-posterior patterns of CNS axons, which connect the brain and the spinal cord.
The invention disclosed herein is based on the discovery of a molecular regulatory system involving Wnt proteins that is involved in the normal formation of the spinal cord axon connection. A chemoattractant gradient exists inside the spinal cord, and this chemoattractant gradient guides the anterior projection of post-crossing spinal cord commissural neurons along the A-P axis towards the brain during embryogensis. In particular, it has been discovered that several Wnt proteins can stimulate the extension of post-crossing but not pre-crossing commissural axons in the spinal cord. Wnt4 was found to be expressed in a decreasing A-P gradient in the floor plate of the spinal cord. sFRPs, inhibitors of Wnts, were found to disrupt the A-P pathfinding of post-crossing spinal cord commissural neurons. However, Wnt4 protein was found to rescue the anterior turn of the misrouting axons and also reorient axons posteriorly, suggesting that Wnt4 plays an instructive role in orienting directional axonal growth. In addition, commissural axons infz3 knockout mice were found to display A-P guidance defects after midline crossing. In view of these findings, Wnt, Wnt-like substances, and/or chemical compounds affecting a Wnt signaling pathway can be used as novel agents to modulate neuronal growth, and can be used in new forms of treatment of diseases and conditions associated with neuronal dysfunction, such as SCI (Lyuksyotova et al., 2003).
The inventor has further found that a different set of Wnt proteins pattern the connections of corticospinal tract (CST) axons projecting along the opposite direction by a repulsive mechanism. CST axons project from the motor cortex of the brain to the spinal cord motor circuits and send voluntary movement signals from the brain to the body. Several Wnt genes were found to be expressed at the dorsal funiculus in an anterior-to-posterior decreasing gradient at the cervical spinal cord, where CST axons first enter the spinal cord and a anterior-to-posterior increasing gradient at the lumbar spinal cord level, forming a “half-pipe” gradient. Wnt1 and Wnt5a can repel CST axons in collagen gel assays. A repulsive Wnt receptor, Ryk (Oshikawa et al., 2003; Halford et al., 2000), is expressed in the CST axons and can be detected at the pyramidal decussation and in the dorsal funiculus. Antibodies against the ectodomain of Ryk can block the repulsion of Wnt1. Finally, intrathecal injection of a Wnt inhibitor, secreted Frizzled related protein 2 (sFRP2), at the rostral cervical level (C1 and C2), can inhibit the posterior growth of CST axons in vivo, leading to weaker grip strength.
The inventor has also found that Wnts play important roles in patterning the synaptic connections once they reach their target. This process of target selection ensures the specific neuron to neuron connection and is essential to the development of the functional circuits throughout the nervous system. Therefore, Wnts can be used to ensure specific synaptic reconnection in repair damaged neural circuits.
Certain embodiments of the present invention are generally concerned with methods for modulating growth of a neuron comprising contacting the neuron with a Wnt, a Wnt-like substance, and/or a chemical compound affecting a Wnt signaling pathway. The definitions of Wnt, Wnt-like substance, and chemical compound affecting a Wnt signaling pathway are discussed in detail in the specification below.
In the context of the invention, the terms “contact” or “contacting” are defined to mean any manner in which a compound is brought into a position where it can mediate, modulate, or inhibit the growth of a neuron. “Contacting” can comprise injecting a diffusable or non-diffusable substance into the neuron or an area adjacent a neuron. “Contacting” can comprise placing a nucleic acid encoding a compound into or close to a neuron or non-neuronal cell in a manner such that the nucleic acid is expressed to make the compound in a manner in which it can act upon the neuron. Those of skill in the art, following the teachings of this specification, will be able to contact neurons with substances in any manner.
The methods for modulating growth of a neuron may, in certain embodiments, be methods for stimulating growth of a neuron, methods for regenerating a damaged neuron, or methods for guiding growth of a neuron along the anterior-posterior axis. In other embodiments, the methods for modulating growth of a neuron are further defined as methods for directionally orienting axon growth of a neuron between the spinal cord and the brain.
The neuron to be modulated may be any neuron. However, in certain embodiments, the neuron is a neuron in the spinal cord that has been damaged. For example, the spinal cord may have been damaged by traumatic spinal cord injury. The damage may have resulted in impaired function of the neuron.
In certain embodiments, the method for modulating growth of a neuron is a method for modulating growth of a neuron in a subject. Although any subject is contemplated by the present invention, in certain embodiments the subject may be a patient with a disorder of the spinal cord. The disorder of the spinal cord may be any disorder, such as a traumatic spinal cord injury. The traumatic spinal cord injury may or may not have resulted in paralysis of the subject. In further embodiments, the patient is a patient with a neurodegenerative disease.
The neuron to be modulated can be a sensory or a motor neuron. In certain embodiments, the neuron is contacted with a Wnt, a Wnt-like substance, and/or a chemical compound affecting a Wnt signaling pathway that further involves exposing the neuron to a gradient of the Wnt, Wnt-like substance, and/or chemical compound affecting a Wnt signaling pathway. The gradient may be in the spinal cord, such as a decreasing anterior-posterior gradient within the spinal cord. In other embodiments, exposing the neuron to the gradient involves stimulating directionally-oriented axon growth of the neuron along the anterior-posterior axis. Any direction of axon growth is contemplated by the present invention. In certain embodiments, the axon growth is directed from the spinal cord to the brain, such as in the growth of neurons in ascending somatosensory pathways. In other embodiments, the axon growth is directed from the brain to the spinal cord, such as in the growth of neurons in descending motor pathways or other regulatory pathways. In further embodiments, the axon growth is directed along the spinothalamic pathway.
Any Wnt is contemplated by the present invention. A detailed discussion of Wnts is provided in the specification below. For example, the Wnt protein may be Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt 6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, or Wnt16 protein. One of skill in the art would be familiar with the range of Wnts available that are contemplated by the present invention. In certain embodiments, the Wnt is a Wnt1, Wnt4, Wnt5a, Wnt6, or Wnt7b protein. In certain embodiments, the Wnt protein will be a mammalian Wnt protein, for example a human or murine Wnt protein, or a homolog thereof from another vertebrate species.
In further embodiments, the Wnt-like substance is a Wnt polypeptide. Any Wnt polypeptide is contemplated by the present invention. For example, the Wnt polypeptide may be a Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt 6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, or Wnt16 polypeptide. One of skill in the art would be familiar with the range of Wnt polypeptides available that are contemplated by the present invention. In certain embodiments, the Wnt polypeptide is a Wnt1, Wnt4, Wnt5a, Wnt6, or Wnt7b polypeptide. Wnt polypeptides are discussed in greater detail in the specification below. In certain embodiments, the Wnt polypeptide will be a mammalian Wnt protein, for example a human or murine Wnt polypeptide, or a homolog thereof from another vertebrate species.
In further embodiments, the Wnt-like substance is a Wnt peptide. Any Wnt peptide is contemplated by the present invention. For example, the Wnt peptide may be a Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt 6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, or Wnt16 peptide. One of skill in the art would be familiar with the range of Wnt peptides available that are contemplated by the present invention. In certain embodiments, the Wnt peptide is a Wnt1, Wnt4, Wnt5a, Wnt6, or Wnt7b peptide. Wnt peptides are discussed in greater detail in the specification below. In certain embodiments, the Wnt protein will be a mammalian Wnt peptide, for example a human or murine Wnt peptide, or a homolog thereof from another vertebrate species.
In other embodiments, the Wnt-like substance is a mimetic of Wnt or a mutant Wnt. The definitions of mimetic Wnt and mutant Wnt are discussed in the specification below. Any Wnt mimetic is contemplated by the present invention. For example, the Wnt mimetic may be a Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt 6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, or Wnt16 mimetic. One of skill in the art would be familiar with the range of Wnt mimetics available that are contemplated by the present invention. In certain embodiments, the Wnt mimetic is a Wnt1, Wnt4, Wnt5a, Wnt6, or Wnt7b mimetic. In certain embodiments, the Wnt mimetic will be a mammalian Wnt mimetic, for example a human or murine Wnt mimetic, or a homolog thereof from another vertebrate species. Any Wnt mutant is contemplated by the present invention. For example, the Wnt mutant may be a Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt 6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, or Wnt16 mutant. One of skill in the art would be familiar with the range of Wnt mutants available that are contemplated by the present invention. In certain embodiments, the Wnt mutant is a Wnt1, Wnt4, Wnt5a, Wnt6, or Wnt7b mutant. In certain embodiments, the Wnt mutant will be a mammalian Wnt mutant, for example a human or murine Wnt mutant, or a homolog thereof from another vertebrate species. In other embodiments, the Wnt-like substance is a small molecule.
Further embodiments of the present invention involve use of chemical compounds affecting a Wnt signaling pathway to modulate growth of a neuron. The definition of such chemical compounds is described in the specification below. One of ordinary skill in the art would be familiar with the wide range of such compounds available which can modulate the Wnt signaling pathway. For example, in certain embodiments, the chemical compound affecting a Wnt signaling pathway is lithium.
The Wnt, Wnt-like substance, and/or chemical compound affecting a Wnt signaling pathway may include a fused amino acid sequence that is designed to facilitate incorporation of the polypeptide into the intracellular compartment of a cell. For example, the Wnt-like substance may include a polypeptide encoding an amino acid TAT sequence from HIV. In another example, the Wnt-like substance may include a polypeptide encoding an Antp amino acid sequence. In another example, the Wnt-like substance may include a polypeptide encoding a VP22 amino acid sequence from HSV.
In certain embodiments, the Wnt, Wnt-like substance, and/or chemical compound affecting a Wnt signaling pathway further includes an expression cassette comprising a promoter, active in a cell, operably linked to a polynucleotide encoding the Wnt, Wnt-like substance, and/or chemical compound affecting a Wnt signaling pathway. For example, the polypeptide may be a Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt 6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, or Wnt16 polypeptide. In certain embodiments, the Wnt polypeptide is a Wnt1, Wnt4, Wnt5a, Wnt6, or Wnt7b polypeptide. In other embodiments, the expression cassette is carried in a viral vector. Although any viral vector is contemplated by the present invention, examples include an adenoviral vector, a retroviral vector, an adeno-associated viral vector, a vaccinia viral vector, or a pox viral vector. In other embodiments, the expression cassette is carried in a nonviral vector, such as a liposome. One of skill in the art would be familiar with a wide range of viral and nonviral vectors available to be of use in the present invention.
Any promoter is contemplated for use in the present invention, as long as it facilitates expression of the polynucleotide. One of skill in the art would be familiar with the wide range of promoters available. For example, the promoter may be a constitutive promoter, an inducible promoter, or a tissue-specific promoter.
Certain embodiments of the present invention involve obtaining the Wnt, Wnt-like substance, and/or chemical compound affecting a Wnt signaling pathway from media of cultured cells. Although any cultured cells are contemplated by the present invention, in certain embodiments the cultured cells comprise an expression cassette including a promoter, active in the cultured cells, operably linked to a polynucleotide encoding Wnt, a Wnt-like substance, and/or a chemical compound affecting a Wnt signaling pathway. The characteristics of expression cassettes that have been previously discussed above apply to these embodiments of the present invention.
Further embodiments of the present invention provide for methods of inhibiting growth of a neuron. In certain embodiments, these methods involve contacting the neuron with a mutant Wnt.
Additional embodiments of the present invention include methods for identifying a substance that modulates growth of a neuron, including: (a) obtaining a candidate substance; (b) contacting said candidate substance with said neuron; and (c) measuring modulation of growth of said neuron. In certain embodiments, an explant assay is used in the methods for identifying a substance that modulates growth of a neuron. For example, the explant assay may involve use of cultured spinal cord. Any method to measure modulation of neuronal growth is contemplated by the present invention. However, in certain embodiments anterior turning of axons of the neuron is measured.
Any candidate substance is contemplated by the present invention. For example, the candidate substance may include a protein, a polypeptide, a peptide, mimetic, mutant, or a small molecule as described above. In a certain embodiments, the candidate substance is a Wnt-like substance, such as a Wnt peptide. Any Wnt peptide is contemplated by the present invention. For example, the Wnt peptide may be a Wnt1 peptide, a Wnt3 peptide, a Wnt4 peptide, a Wnt5a peptide, a Wnt6 peptide, or a Wnt7b peptide. In certain embodiments, the Wnt peptide is a mimetic of Wnt, such as a mimetic of Wnt1, a mimetic of Wnt3, a mimetic of Wnt4, a mimetic of Wnt5a, a mimetic of Wnt6, or a mimetic of Wnt7b. In a further embodiment, the Wnt-like substance is a mimetic of Wnt4. Alternatively, the Wnt-like substance may be a mutant Wnt, such as a mutant Wnt1 polypeptide, a mutant Wnt3 polypeptide, a mutant Wnt4 polypeptide, a mutant Wnt5a polypeptide, a mutant Wnt6 polypeptide, or a mutant Wnt7b polypeptide. In still further embodiments, the Wnt-like substance is a small molecule. In other embodiments, the chemical compound affecting a Wnt signaling pathway is a chemical compound that functionally or structurally resembles lithium.
Any method of measuring growth of a neuron is contemplated by the present methods for identifying modulators of nerve growth. These methods have been discussed above. For example, measuring modulation of growth of a neuron may further involve measuring stimulation of growth of the neuron, measuring regeneration of a damaged neuron, or measuring growth of said neuron along the anterior-posterior axis. In addition, these methods also involve method for directionally orienting axon growth of the neuron between the spinal cord and the brain.
The present invention also includes methods of modulating growth of a neuron in a subject, including: (a) providing a composition that includes a Wnt, a Wnt-like substance, and/or a chemical compound affecting a Wnt signaling pathway; and a pharmaceutical preparation suitable for delivery to the subject; and (b) administering the composition to the subject. The methods for modulating neuron growth of the present invention contemplate measurement of neuronal growth by any known means, as discussed above. For example, the method of modulating neuron growth may be defined as a method of promoting growth and regeneration of a neuron in a subject, a method of promoting axon growth and regeneration in a subject, or a method of promoting directionally-oriented axon growth in a subject. Directionally-oriented axon growth may be along the anterior-posterior axis such as from the spinal cord to the brain, or from the brain to the spinal cord.
The methods for modulating neuron growth in a subject contemplated by the present invention also include methods of treating a subject with a spinal cord disorder. Any spinal cord disorder is contemplated by the present invention. For example, the spinal cord disorder may be a traumatic spinal cord disorder, a disorder of motor and/or sensory neurons, a neurodegenerative disorder, or a disorder resulting in paralysis.
The methods of the present invention also contemplate exposing the neuron to a gradient of said Wnt, said Wnt-like substance, and/or said chemical compound affecting a Wnt signaling pathway. As discussed above, the gradient may be in the spinal cord, such as a decreasing gradient along the anterior-posterior axis.
Any Wnt, Wnt-like substance, and chemical compound affecting a Wnt signaling pathway, as discussed above and in the specification below, is contemplated by the present methods of modulating neuron growth in a subject. Mimetics and mutants of Wnts and Wnt-like substances are contemplated by the present invention, as are embodiments wherein the Wnt or Wnt-like substance further comprises an expression cassette comprising a promoter, active in a cell, operably linked to a polynucleotide encoding the Wnt or the Wnt-like substance. These expression cassettes have been discussed above, and are discussed in greater detail in later sections of this specification.
In certain embodiments, administering the composition of Wnt, Wnt-like substance, and/or chemical compound affecting a Wnt signaling pathway involves contacting the composition with the spinal cord of the subject. In certain embodiments, a gradient of the Wnt, Wnt-like substance, and/or chemical compound affecting a Wnt signaling pathway is created along the anterior-posterior axis. For example, the gradient may be between the spinal cord and the brain, such as a decreasing anterior-posterior gradient. In certain embodiments, the nerve cell is contacted with a modulator of neuronal growth identified by one of the previously described methods.
Certain embodiments of the present invention pertain to pharmaceutical compositions for modulating growth of a neuron in a mammal, including: (a) a Wnt, a Wnt-like substance, and/or a chemical compound affecting a Wnt signaling pathway; and (b) a pharmaceutical preparation suitable for delivery to the mammal. Neuronal growth may be modulated by any of the methods discussed above. In certain embodiments, the mammal is a human, such as a patient with a spinal cord disorder. Any Wnt, Wnt-like substance, and/or chemical compound affecting a Wnt-signaling pathway, as discussed above, is contemplated by the present invention. In certain embodiments, the composition comprises an expression cassette comprising a promoter, active in a cell, operably linked to a polynucleotide encoding the Wnt, the Wnt-like substance, and/or the chemical compound affecting a Wnt signaling pathway. Expression cassettes have been discussed above in the context of other embodiments of the present invention.
Additional embodiments of the present invention involve methods of inhibiting or controlling the growth of a neuron in a subject, by administering an inhibitor of a Wnt to the subject. In some cases, that inhibitor may be an sFRP, a Ryk protein, or an analog thereof. In general some such methods include: (a) providing a composition that includes an sFRP, an sFRP-like substance, a Ryk or a Ryk-like substance and a pharmaceutical preparation suitable for delivery to the subject; and (b) administering said composition to the subject. sFRPs are compounds that can affect a Wnt signaling pathway by binding to Wnt proteins with high affinity and blocking the interaction of Wnts with their receptors, the Frizzleds. sFRPs and sFRP-like substances are defined and discussed in detail below.
In certain embodiments, the composition comprises an sFRP protein. sFRPs are diffusable proteins that bind and modulate Wnts. Any sFRP protein from any species is contemplated by the present invention. For example, the sFRP protein may be sFRP1 protein, sFRP2 protein, or sFRP3 protein. In other embodiments, the sFRP-like substance is an sFRP polypeptide. For example, the sFRP polypeptide may be sFRP1 polypeptide, sFRP2 polypeptide, or sFRP3 polypeptide. In other embodiments, the sFRP-like substance is a peptide, such as sFRP1 peptide, sFRP2 peptide, or sFRP3 peptide. In further embodiments, the sFRP-like substance is a mutant sFRP, such as a mutant sFRP1 polypeptide, a mutant sFRP2 polypeptide, or a mutant sFRP3 polypeptide. In still further embodiments, the sFRP-like substance includes a small molecule that is functionally similar to a sFRP.
In other embodiments, the composition comprises a Ryk protein. Ryk is a receptor on neurons that binds Wnts and mediates repulsion of neurons in response to Wnts. Any Ryk protein or homolog from any species is contemplated by the present invention, for example, Drosphila Derailed protein may be employed in some embodiments. For example, the Ryk or Ryk-like substance may be a Ryk protein, polypeptide, peptide, mutant, or mimetic. In still further embodiments, the Ryk-like substance includes a small molecule that is functionally similar to a Ryk.
Other embodiments of the invention involve the contacting of a neuron with a combination of a Wnt and another substance, in order to provide a combination therapy. Such embodiments of the invention are important because, as discussed herein, the regeneration of neurons into a properly functioning spinal cord will often involve a combination of directional and other clues.
In some embodiments, one will wish to contact a neuron with a substance that blocks activity of a neuronal growth inhibitor. Such neuronal growth inhibitors include the myelin inhibitors Nogo, MAG, and Omgp, which have been shown to inhibit the growth of sensory neurons. Further, as discussed herein, Wnts can, if expressed in the adult spinal cord, inhibit the proper growth of CST motor neurons. In this regard, there are some Wnts that are expressed in normal adult spinal cords, and a variety of Wnts that may be is abnormally expressed in the neuron upon neuronal injury, as discussed below. In some embodiments of the invention, the substance that blocks the activity of the neuronal growth inhibitor is an antibody directed against a receptor for the inhibitor on the neuron or against the inhibitor itself. For example, such an antibody can be directed against a Wnt, Nogo, MAG, or OMgp. In some preferred embodiments, the antibody is directed against Wnt5a, Wnt8, or a Wnt that is expressed abnormally in the neuron due to injury, or against a receptor of any such Wnt. In other cases, the substance that blocks activity of a neuronal growth inhibitor is a Ryk, Ryk-like substance, sFRP or sFRP-like substance. In some preferred embodiments, one will want to block the activity of two or more inhibitors in the course of treating a neuron, spinal cord, and/or patient. For example, in order to allow an injured spinal cord comprising both injured sensory and injured motor neurons to regenerate in an appropriate manner, those of skill will understand that there may be a need to apply a compound to block the myelin inhibitors and prevent them from inhibiting the growth of sensory neurons, while also applying a compound to block Wnt inhibition of the growth of motor neurons.
The instant invention also involves contacting neurons with combinations of at least one Wnt and at least one other substance that attracts or repels neuronal growth. In some embodiments, the at least one other substance will be a substance attracts neuronal growth, for example, but not limited to a Wnt, Netrin, Shh, Cell adhesion molecule, Ig superfamily member, Cadherin, Integrin, EphrinB, ECM molecule, or HGF. In some embodiments, the at least one other substance will be a substance that repels neuronal growth, for example but not limited to, a Semaphorins, Netrin, Slit, Wnt, BMP, Ephrin, or member of the Ig superfamily. In many embodiments, contacting said neuron with a substance that attracts or repels neuronal growth will comprise exposing said neuron to a gradient of said substance. And, in some embodiments, the neuron will be exposed to a gradient of at least two such substances. In some cases, it will be beneficial to apply inhibitors of these substances that attract or repel neuronal growth at various portions of a regenerating spinal cord, in order to control the growth of the spinal cord, such inhibitors can be small molecules, peptides, proteins, or polypeptides that bind the substance, antibodies directed against the substance or a receptor of the substance, etc.
Some embodiments will involve the exposure of the neuron to a gradient of an attractive Wnt, some will involve exposure of the neuron to a gradient of a repulsive Wnt, some will involve exposure of the neuron to gradients of both attractive and repulsive Wnts. Attractive Wnts can include, but not be limited to, Wnt1, Wnt4, Wnt5a, Wnt 6, and Wnt7. Repulsive Wnts can include, but not be limited to Wnt5a or Wnt1. Those of skill in the art will be able to determine attractive and repulsive Wnts following the teachings herein, and will understand that the same Wnt may have an attractive property in regard to some contexts or some types of neurons and a repulsive property in regard to other contexts or types of neurons.
In some cases, it will be beneficial to apply one or more Wnt to the site of a spinal cord injury, such that the Wnt(s) will provide attractive guidance to those neurons that need to be attracted to the site of injury during regeneration and repellant guidance to those neurons that need to grow away from the site of injury during regeneration. In this regard, Wnt(s) applied at the site of an injury will provide directional guidance to axonal growth and cause sensory neurons to grow up through the site of the injury and repel motor neurons to grow down through the site of the injury. Further, in this embodiment, it may be beneficial to inhibit the Ryk pathway at the site of the injury so that motor neurons growing through the site of the injury are not inhibited by any Wnts present in the injury site, whether those Wnts are applied to the injury site, or expressed there as a result of normal adult Wnt expression or injury-induced Wnt expression. One may also apply a blocker of myelin inhibitors to the injury site, to prevent such inhibitors from impacting the growth of sensory neurons through the site.
Of course, combinations of Wnts, substances that block inhibitors of neuronal growth, and/or substances that attract or repel neuronal growth can be determined by those of skill in the art following the teaching contained herein. These various components of these combinations may be administered simultaneously, or separated by time. Individual components may be administered a single time or in a series of administrations. They may be administered in a single pharmaceutical composition, or in separate compositions. Those of skill in the art will be able to follow the teachings of this specification to determine appropriate dosage regimes and schedules of the various active agents.
Other embodiments of the invention involve pharmaceutical compositions comprising at least one Wnt, Wnt-like substance, or compound affecting a Wnt signaling pathway in combination with at least one substance that blocks an inhibitor of neuronal growth, and/or substance that attracts or repels neuronal growth. Further, kits comprising combinations of these various components, in separate or single containers are also within the scope of the invention.
As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more.
The term “therapeutically effective” as used herein refers to an amount of a compound required to effect neuronal growth in the context of the manners described herein.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
The present invention is based on the discovery that Wnts guide axon pathfinding in development and can play a role in correct spinal cord and neuronal regeneration.
The inventor has shown that a Wnt/Frizzled pathway mediates attractive effects in sensory axon guidance along the anterior-posterior axis. Additionally, the inventor shows here that vertebrate corticospinal cord axons are repelled by Wnts and the repulsion is mediated by the vertebrate homologue of Derailed, Ryk. Ryk is not expressed in the commissural neurons, consistent with the finding that commissural axons are attracted by Wnts. Interestingly, the repulsive effect of Wnt5 on fly axons appears to be independent of Frizzleds. Therefore, Wnts appear to attract axons via a Frizzled-dependent pathway and repel axons via a Ryk dependent pathway. CST axons do express Frizzleds, such as Frizzled 3. Therefore, it appears that Ryk is dominate over Frizzleds and mediates repulsion even in the presence of Frizzleds. Taken together, these studies provide evidence that Wnts, like other guidance cues, are bifunctional, capable of attracting some axons and repelling others, and suggest that Wnt proteins might have a widespread and phylogenetically conserved function in guiding axons during the wiring of the nervous system. These studies demonstrate that one continuous molecular gradient of diffusible guidance cue(s) along the entire anterior-posterior axis of the spinal cord controls the navigation decisions along the A-P axis.
The present invention seeks to exploit the inventor's discovery by providing for methods and compositions for modulating growth of a nerve cell using a Wnt, Wnt-like substances, and/or chemical compounds to stimulate the pathways of Wnt signaling to modulate nerve growth and guidance. These methods and compositions can be used in a wide variety of therapeutic contexts where nerve growth and regeneration would be beneficial. For example, a Wnt, a Wnt-like substance, and/or a chemical compound affecting a Wnt signaling pathway can be used to stimulate axonal growth of a damaged neuron along the A-P axis of a patient with SCI. Because it has also been observed that the Wnts are expressed in the several regions in the brain and the components of the Wnt signaling pathways are also present in axons of other central nervous system neurons, it is possible that Wnts and agents that stimulate or inhibit Wnt signaling can be used to modulate growth and directional guidance of axons in the central nervous system.
A. Wnt, Wnt-Like Substances, and Compounds Affecting a Wnt Signaling Pathway1. Wnt and Wnt-Like Substances
The present invention pertains to use of Wnt and Wnt-like substances in various contexts. For example, various embodiments of the present invention pertain to methods for modulating growth of a neuron that involve contacting a neuron with a Wnt or a Wnt-like substance. Other embodiments pertain to methods for modulating growth of a neuron in a subject, that involve providing the subject with a pharmaceutical composition that includes a Wnt or a Wnt-like substance. Additional embodiments pertain to pharmaceutical compositions for modulating growth of a neuron in a mammal, that include a Wnt or a Wnt-like substance.
As discussed above, Wnts are secreted cysteine-rich glycosylated proteins that play a role in the development of a wide range of organisms. Wnts are thought to function in a variety of developmental and physiological processes since many diverse species have multiple conserved Wnt genes (McMahon, 1992; Nusse and Varmus, 1992). The Wnt growth factor family includes at least 19 genes identified in mammals, including Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt 6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16. Similar numbers of Wnt genes are present in other vertebrate species. Of course, further Wnts may be discovered and/or characterized in the future, and those of skill will be able to employ any such Wnts in the context of the invention. Further, those of skill will be able to use the teachings herein to obtain and use Wnts of any species in the context of the invention.
Throughout this application, the term “Wnt” is intended to refer to any consecutive amino acid sequence that includes the full-length amino acid sequence of a Wnt from any organism, such as a human or a mouse Wnt. Wnt can be a human Wnt protein, or a Wnt protein from any other species, such as mouse or chick. Thus, for example, Wnt can be used to refer to the full-length amino acid sequence encoded by any of the 19 genes identified in human. Alternatively, Wnt can refer to a murine Wnt protein, such as murine Wnt4. Wnt can also refer to an amino acid sequence that is longer than the full-length consecutive amino acid sequence of a Wnt, as long as it includes a full-length Wnt amino acid sequence.
Throughout this application, the term “Wnt protein” is intended to refer to the full-length amino acid sequence that is encoded by a Wnt gene. Thus, “Wnt” may refer to a Wnt protein or an amino acid sequence that is longer than a Wnt protein if additional non-Wnt amino acids are included in the sequence. Also included in the definition of “Wnt” is a truncated sequence of a Wnt protein, a mutated Wnt protein, or a Wnt amino acid sequence that is less than the full-length amino acid sequence of a Wnt, as long as the amino acid sequence retains an acceptable level of the equivalent biological activity of a full-length Wnt protein.
The human and murine full-length native amino acid sequences and the native nucleic acids encoding them are described by GenBank accession number in the Table 1. Further, summary of human and murine Wnts is provided in Miller, 2001. Specifically, Table 1 of Miller, 2001, which includes Genbank accession numbers of human and mouse Wnt genes, is herein specifically incorporated by reference.
Throughout this application, the term “Wnt-like substance” is intended to refer to a Wnt polypeptide, a Wnt peptide, a Wnt mimetic, or a small molecule that is functionally and/or structurally similar to a Wnt.
The term “Wnt polypeptide” includes any amino acid sequence that includes fewer consecutive amino acids of a Wnt than the full-length amino acid sequence of a Wnt. “Wnt polypeptide” includes not only consecutive amino acid sequences from a human Wnt, but from any other species, such as mouse. Thus, for example, a Wnt polypeptide can include, but is not limited to, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or greater amino molecule residues of a Wnt, and any range derivable therein, as long as the amino acid sequence includes less than the full-length consecutive amino acid sequence of a Wnt. Included within the definition of “Wnt polypeptide” are potential amino acid sequences that include additional amino acids, other than Wnt amino acid sequences.
The term “Wnt peptide” includes any amino acid sequence that includes ten or fewer consecutive amino acid sequence of a Wnt amino acid sequence. “Wnt peptide” includes not only consecutive amino acid sequences from a human Wnt, but from any other species, such as mouse. Thus, for example, a Wnt peptide may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids of a Wnt. Additional amino acids can also be included, which may be other than Wnt amino acid sequences.
Included within the definition of “Wnt-like substance” is a “mimetic of Wnt.” Throughout this application, “mimetic of Wnt” is intended to refer to any molecule other than the full-length sequence of a Wnt that is able to maintain an acceptable level of equivalent biological activity as a Wnt.
It is well understood by the skilled artisan that, inherent in the definition of a “mimetic of Wnt,” is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity, e.g., ability of Wnt4 to modulate neuronal growth and regeneration. “Mimetic of Wnt” is thus defined herein as any Wnt polypeptide in which some, or most, of the amino acids may be substituted so long as the polypeptide retains substantially similar activity in the context of the uses set forth herein. Of course, a plurality of distinct proteins/polypeptides/peptides with different substitutions may easily be made and used in accordance with the invention. Additionally, in the context of the invention, a mimetic of Wnt can be a Wnt homologue polypeptide from any species or organism, including, but not limited to, a human polypeptide. One of ordinary skill in the art will understand that many mimetics of Wnt would likely exist and can be identified using commonly available techniques.
The present invention may utilize Wnts, Wnt polypeptides, Wnt peptides, mimetics of Wnt, or mutants of Wnt, that are purified from a natural source or from recombinantly-produced material. Those of ordinary skill in the art would know how to produce these amino acid sequences from recombinantly-produced material. This material may use the 20 common amino acids in naturally synthesized proteins, or one or more modified or unusual amino acids. Generally, “purified” will refer to an Wnt composition that has been subjected to fractionation to remove various other proteins, polypeptides, or peptides, and which composition substantially retains its activity. Purification may be substantial, in which the Wnt or Wnt-like substance is the predominant species, or to homogeneity, which purification level would permit accurate degradative sequencing.
Amino acid sequence mutants of a Wnt also are encompassed by the present invention, and are included within the definition of “Wnt-like substance.” Amino acid sequence mutants of a Wnt of any species, such as human and mouse Wnt, is contemplated by the present invention. Amino acid sequence mutants of a Wnt can be substitutional mutants or insertional mutants. Insertional mutants typically involve the addition of material at a non-terminal point in the peptide. This may include the insertion of a few residues; an immunoreactive epitope; or simply a single residue. The added material may be modified, such as by methylation, acetylation, and the like. Alternatively, additional residues may be added to the N-terminal or C-terminal ends of the peptide.
Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, or example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents.
Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, or example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents.
In making changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated by reference herein). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within +2 is preferred, those which are within +1 are particularly preferred, and those within +0.5 are even more particularly preferred.
It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent protein. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5+1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).
In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within +2 is preferred, those which are within +1 are particularly preferred, and those within +0.5 are even more particularly preferred.
Certain embodiments of the present invention utilize Wnt-like substances that are fusion proteins that are preferentially translocated through biological membranes. In particular, a Wnt or a Wnt-like substance such as a Wnt polypeptide may be fused to a particular protein, polypeptide, or peptide sequence that promotes facilitated intracellular delivery of the fusion protein into the targeted cell. Although any fusion protein with the property of facilitated intracellular delivery is contemplated by the present invention, specific examples include fusion proteins utilizing the HIV TAT sequence (Nagahara et al., 1998), the third helix of the Antennapedia homeodomain (Antp) (Derossi et al., 1994), and the HSV-1 structural protein VP22 (Elliott and O'Hare, 1997).
Small molecules are also included within the definition of “Wnt-like substance” in the context of the present invention. Throughout this application, the term “small molecule” is intended to refer to any small molecule not included within the definition of Wnt polypeptide, Wnt peptide, mimetic of Wnt, or mutant of Wnt, wherein the molecule is relatively small in size and wherein the molecule has an acceptable level of biological activity of a Wnt. For example, the small molecule may be a synthetic substance which is not an amino acid sequence, which is functionally able to promote axonal growth and regeneration in a manner analogous to a Wnt.
2. Polynucleotides Encoding a Wnt or a Wnt-Like Substance
Various aspects of the present invention require polynucleotides encoding an Wnt or a Wnt-like substance. For example, various embodiments include methods for modulating neuronal growth that involve contacting the neuron with an expression cassette that includes a promoter that is a cell, operably linked to a polynucleotide encoding either an Wnt or a Wnt-like substance. In other embodiments, the invention pertains to methods for modulating growth of a neuron in a subject that include administering to the subject a composition that includes an expression cassette operably inked to a polynucleotide encoding either a Wnt or a Wnt-like substance. In still other embodiments, the invention includes pharmaceutical compositions for modulating growth of a neuron in a mammal, that include a Wnt or a Wnt-like substance.
The polynucleotide encoding the full length amino acid sequences of the known human and murine Wnts are contained in Table 1. The polynucleotides according to the present invention may encode an entire Wnt sequence (e.g., the amino acid sequence of SEQ ID NO:2), or a Wnt-like substance such as a Wnt polypeptide or a Wnt peptide. The polynucleotides may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism.
In other embodiments, however, the polynucleotides may be complementary DNA (cDNA). cDNA is DNA prepared using messenger RNA (mRNA) as a template. Thus, a cDNA does not contain any interrupted coding sequences and usually contains almost exclusively the coding region(s) for the corresponding protein. In other embodiments, the polynucleotide may be produced synthetically.
It may be advantageous to combine portions of the genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used. Introns may be derived from other genes in addition to a Wnt gene. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
The present invention is not limited to the sequences disclosed by GenBank and SEQ ID NO in Table 1, but includes polynucleotides encoding any Wnt or Wnt-like substance (discussed above). These polynucleotides encoding a Wnt or a Wnt-like substance may be naturally-occurring homologous polynucleotide sequences from other organisms. For example, polynucleotides encoding a Wnt or a Wnt-like substance include those polynucleotides encoding the human amino acid functional equivalent sequences previously described. These sequences are provided by way of example, and are not meant to be a summary of all available polynucleotide sequences encoding a Wnt or a Wnt-like substance. A person of ordinary skill in the art would understand that commonly available experimental techniques can be used to identify or synthesize polynucleotides encoding other Wnts. The present invention also encompasses chemically synthesized mutants of these sequences.
Another kind of sequence variant results from codon variation. Because there are several codons for most of the 20 normal amino acids, many different DNAs can encode a Wnt or a Wnt-like substance. Reference to the following table will allow such variants to be identified.
Allowing for the degeneracy of the genetic code, sequences that have between about 50% and about 75%, or between about 76% and about 99%, of nucleotides that are identical to the nucleotides disclosed herein will be preferred. Sequences that are within the scope of “a polynucleotide encoding a Wnt or a Wnt-like substance” are those that are capable of base-pairing with a polynucleotide segment set forth above under intracellular conditions.
As stated above, the encoding sequences may be full length genomic or cDNA copies, or large fragments thereof. The present invention also may employ shorter oligonucleotides. Sequences of 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence. Although shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of base-pairing. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 base pairs will be used, for example, in the preparation of mutants of Wnt and in PCR reactions.
Any sequence of 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence. Although shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of hybridization. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length.
In certain embodiments, one may wish to employ constructs which include other elements, for example, those which include C-5 propyne pyrimidines. Oligonucleotides which contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity (Wagner et al., 1993).
3. Compounds that can Affect the Wnt Signaling Pathways
a. Chemical Compounds that can Affect the Wnt Signaling Pathway
As an alternative approach to using the a Wnt or a Wnt-like substance to directly modulate axon growth and guidance to promote axonal regeneration to cure spinal cord injury and other central nervous system damage, chemical compounds which affect the Wnt signaling pathways and affect axonal regeneration can also be applied to promote and guidance axon regeneration. Such chemical compound can be discovered by “chemical genetics”, screening libraries of chemical compounds or testing known compounds that have an effect on Wnt signaling. For example, lithium is known to stimulate Wnt signaling and can promote axon extension (Hall et al., 2000; Klein and Melton, 1996; Lucas and Salinas, 1997). Therefore, chemical substances, such as lithium, can be used to regulate the Wnt pathway and help regenerate spinal cord axons and other central nervous system axons.
b. sFRPS can Affect the Wnt Signaling Pathways
Secreted Frizzled-related proteins (sFRPs) are soluble proteins that can bind to Wnt proteins with high affinities and can block the interaction of Wnts with their receptors, the Frizzleds (Wodarz and Nusse, 1998). Any sFRP, whether from human or any other species such as mouse, is contemplated by the present invention. In addition, the definition of sFRP-like substance is defined in a similar manner as Wnt-like substance, and includes mimetics of sFRP and mutant sFRPs.
The definition of sFRP, sFRP-like substance, sFRP protein, and sFRP polypeptide are defined in a manner analogous to the definitions provided above in reference to Wnt and Wnt-like substance, discussed supra.
The full-length amino acid sequence of human sFRP1 (Genbank accession number NP—003003) is provided herein as SEQ ID NO:77. The full-length amino acid sequence of human sFRP2 (Genbank accession number XP—050625) is provided herein as SEQ ID NO:78. The full-length amino acid sequence of human sFRP3 (Genbank accession number NP—001454) is provided herein as SEQ ID NO:79. The full-length amino acid sequence of murine sFRP1 (Genbank accession number NP—038862) is provided herein as SEQ ID NO:80. The full-length amino acid sequence of murine sFRP2 (Genbank accession number NP—033170) is provided herein as SEQ ID NO:81. The full-length amino acid sequence of murine sFRP3 (Genbank accession number AAC53147) is provided herein as SEQ ID NO:82.
c. Ryk can Affect the Wnt Signaling Pathways
Ryk is a protein that can bind to Wnt proteins with high affinities and can block the activity of at least some of Wnts. Ryk is a vertebrate homolog of the Drosphila Derailed protein, a receptor tyrosine-like protein. Any Ryk, whether from human or any other species such as mouse, is contemplated by the present invention. In addition, the definition of Ryk-like substance is defined in a similar manner as Wnt-like substance, and includes mimetics of Ryk and mutant Ryks.
The definition of Ryk, Ryk-like substance, Ryk protein, and Ryk polypeptide are defined in a manner analogous to the definitions provided above in reference to Wnt and Wnt-like substance, discussed supra.
The full-length amino acid sequence of human Ryk (Genbank accession number NM—002958) is provided herein as SEQ ID NO:83. The full-length amino acid sequence of murine Ryk (Genbank accession number BC—006963) is provided herein as SEQ ID NO:84. The full-length amino acid sequence of Derailed (Genbank accession number L47260) is provided herein as SEQ ID NO:85.
B. Inhibitors of Axonal GrowthThe adult central nervous system is a largely inhibitory environment for axonal growth and regeneration. Therefore, in the context of obtaining regeneration of the CNS, it is likely that the blocking of such inhibitors will be needed.
Additionally, multiple inhibitors present in the central nervous system myelin, such as Nogo, MAG and OMgp, prevent axonal growth after injury. Other inhibitors present in glial scar, such as CSPG, also inhibit axonal outgrowth. It is not fully understood whether CSPG are the actual active components for the inhibitors of axonal regeneration or other molecules associate with CSPG are the active components.
In order to achieve effective axonal regeneration following CNS injury, it is necessary to overcome inhibition of both type of inhibitors. Those of skill in the art will understand that there are many manners in which such inhibitors can be blocked, and will, by following the teachings contained herein, be able to develop means to block these inhibitors in the context of the invention.
C. Protein Attractants and Repellants in Axonal GuidanceThere are many protein attractants and repellants that play a role in axonal guidance. Further, many such axon guidance molecules are bi-functional: attractive to one type of axons and repulsive to another, depending on the receptor composition in the responding growth cones.
A number of molecules direct axonal growth during development. These compounds are play important roles in embryonic development, and may function in the same or a similar way in the adult CNS.
Attractants and repellants can be divided into two general categories, diffusable and non-diffusable. Diffusible attractants include, but are not limited to, Netrins, Shh, Wnts, and HGF. Diffusible repellents include, but are not limited to, Secreted Semaphorins, Netrins, Slits, Wnts, and BMPs. Non-diffusible attractants include, but are not limited to: cell adhesion molecules such as members of the Ig superfamily, Cadherins, and Integrins; Ephrins; and ECM molecules. Non-diffusable repellents include, but are not limited to, Ephrins, members of the Ig superfamily, and membrane-bound Semaphorins.
Those of skill in the art will be able to use these, and any other attractants or repellants in the context of the invention. For example, those of skill in the are will be able to use these attractants or repellants to create suitable gradients for guiding neuronal growth.
In the context of the invention, native attractants or repellants may be employed. Further, proteins, polypeptides, peptides, mutants, and/or mimetics of these attractants or repellants may be employed, with the definitions of these provided above in reference to Wnt and Wnt-like substance, discussed supra.
D. Targeted Diseases and ConditionsThe present invention contemplates methods of treating a subject that includes administering to the subject a composition that includes a Wnt, a Wnt-like substance, and/or a chemical compound affecting a Wnt signaling pathway in a pharmaceutical preparation suitable for delivery to the subject. Other axonal guidance molecules or substances that block neuronal inhibitors can be administered in combination. The subject can be a patient with a disease wherein neuronal dysfunction plays a prominent role in the pathophysiology. For example, the patient may have a disorder of the spinal cord. Any disorder of the spinal cord is contemplated by the present invention. In certain embodiments, the disorder of the spinal cord is traumatic spinal cord injury (discussed above). The traumatic spinal cord injury may or may not have resulted in paralysis of the subject. The neuronal dysfunction can be by any mechanism. For example, cell death can be the result of acute traumatic injury or degeneration.
In certain embodiments, the Wnt, Wnt-like substance, and/or a chemical compound affecting the Wnt signaling pathway is administered to a subject for the purpose of stimulating and promoting directed axonal growth and regeneration along the anterior-posterior axis of the spinal cord.
Any disease or condition wherein there is neuronal dysfunction is contemplated by the present invention. In addition to SCI, other examples include Parkinson's disease, where dopaminergic neurons undergo degeneration and ALS where neurons in the motor systems undergo degeneration. In these cases, stem cells are being developed so that they can be transplanted to the midbrain and the spinal cord, respectively, so that they can populate and make proper connection with their targets. The establishment of new connections require the directly growth of axons from these neural stem cells. Wnt and Wnt-like substances and other chemical compounds affecting a Wnt signaling pathway can be used in growth and guidance of regenerating axons from these stem cells.
E. Nucleic Acids1. Overview
Certain embodiments of the invention pertain to methods utilizing compositions that include an nucleic acids. In particular, the methods for modulating growth of a neuron may involve contacting the neuron with a Wnt, a Wnt-like substance, and/or a chemical compound affecting a Wnt signaling pathway that further includes an expression cassette. The methods of treating a subject may involve administering to the subject a composition of a Wnt, a Wnt-like substance, and/or a chemical compound affecting a Wnt signaling pathway that includes an expression cassette. One of skill in the art would understand the techniques relating to use of expression cassettes to deliver polynucleotide sequences to cells or subjects. Particular aspects of these techniques of these techniques are summarized in this specification. This brief summary is in no way designed to be an exhaustive overview of all available experimental techniques related to expression cassettes since one of skill in the art would already be familiar with these techniques.
Throughout this application, the term “expression cassette” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein or polypeptide, but it need not be. Thus, in certain embodiments, expression includes both transcription of a gene and translation of a mRNA into a polypeptide.
In order for the expression cassette to effect expression of a polypeptide, the polynucleotide encoding the polynucleotide will be under the transcriptional control of a promoter. A “promoter” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrase “operatively linked” means that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. One of skill in the art would understand how to use a promoter or enhancer to promote expression of a Wnt, a Wnt-like substance, and/or a chemical compound affecting a Wnt signaling pathway.
In certain embodiments of the invention, the delivery of an expression cassette in a cell may be identified in vitro or in vivo by including a marker in the expression vector. The marker would result in an identifiable change to the transfected cell permitting easy identification of expression. The selectable marker employed is not believed to be important, so long as it is capable of being expressed along with the polynucleotide of the expression cassette. Examples of selectable markers are well known to one of skill in the art.
A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals.
In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5′ methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). One of skill in the art would be familiar with use of IRES in expression cassettes.
Expression cassettes can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector. See Carbonelli et al. (1999); Levenson et al. (1998); Cocea (1997). “Restriction enzyme digestion” refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.
In expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed. One of skill in the art would understand how to use these signals to effect proper polyadenylation of the transcript.
In certain embodiments of the present invention, the expression cassette comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis and, in some cases, integrate into the host cell chromosomes, have made them attractive candidates for gene transfer in to mammalian cells. However, because it has been demonstrated that direct uptake of naked DNA, as well as receptor-mediated uptake of DNA complexes, expression vectors need not be viral but, instead, may be any plasmid, cosmid or phage construct that is capable of supporting expression of encoded genes in mammalian cells, such as pUC or Bluescript™ plasmid series. One of ordinary skill in the art would be familiar with use of viruses as tools to promote expression of the polypeptide.
In certain embodiments of the invention, a treated cell may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.
Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is calorimetric analysis, are also contemplated. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable and screenable markers are well known to one of skill in the art.
F. Gene Transfer1. Viral Vectors
In certain embodiments, the methods and compositions of the invention utilize expression cassette which includes a polynucleotide encoding a Wnt, a Wnt-like substance, a chemical compound affecting a Wnt signaling pathway, another axonal guidance molecule, and/or substance that blocks a neuronal inhibitor can be administered in combination, carried in a vector. One of ordinary skill in the art would understand use of vectors since these experimental methods are well-known in the art. In particular, techniques using “viral vectors” are well-known in the art. A viral vector is meant to include those constructs containing viral sequences sufficient to (a) support packaging of the expression cassette and (b) to ultimately express a recombinant gene construct that has been cloned therein.
One method for delivery of the recombinant DNA involves the use of an adenovirus expression vector. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors.
Adenoviruses are currently the most commonly used vector for gene transfer in clinical settings. Among the advantages of these viruses is that they are efficient at gene delivery to both nondividing an dividing cells and can be produced in large quantities. The vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization or adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus et al., 1992). In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.
Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range and high infectivity. A person of ordinary skill in the art would be familiar with experimental methods using adenoviral vectors.
The adenovirus vector may be replication defective, or at least conditionally defective, and the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
Adenovirus growth and manipulation is known to those of skill in the art, and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 109-1011 plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al., 1963; Top et al., 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5′ and 3′ ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).
In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. A person of ordinary skill in the art would be familiar with well-known techniques that are available to construct a retroviral vector.
Adeno-associated virus (AAV) is an attractive vector system for use in the present invention as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells in tissue culture (Muzyczka, 1992). AAV has a broad host range for infectivity (Tratschin, et al., 1984; Laughlin, et al., 1986; Lebkowski, et al., 1988; McLaughlin, et al., 1988), which means it is applicable for use with the present invention. Details concerning the generation and use of rAAV vectors are described in U.S. Pat. No. 5,139,941 and U.S. Pat. No. 4,797,368, each incorporated herein by reference.
AAV is a dependent parvovirus in that it requires coinfection with another virus (either adenovirus or a member of the herpes virus family) to undergo a productive infection in cultured cells (Muzyczka, 1992). In the absence of coinfection with helper virus, the wild-type AAV genome integrates through its ends into human chromosome 19 where it resides in a latent state as a provirus (Kotin et al., 1990; Samulski et al., 1991). rAAV, however, is not restricted to chromosome 19 for integration unless the AAV Rep protein is also expressed (Shelling and Smith, 1994). When a cell carrying an AAV provirus is superinfected with a helper virus, the AAV genome is “rescued” from the chromosome or from a recombinant plasmid, and a normal productive infection is established (Samulski et al., 1989; McLaughlin et al., 1988; Kotin et al., 1990; Muzyczka, 1992).
Typically, recombinant AAV (rAAV) virus is made by cotransfecting a plasmid containing the gene of interest flanked by the two AAV terminal repeats (McLaughlin et al., 1988; Samulski et al., 1989; each incorporated herein by reference) and an expression plasmid containing the wild-type AAV coding sequences without the terminal repeats, for example pIM45 (McCarty et al., 1991; incorporated herein by reference). A person of ordinary skill in the art would be familiar with techniques available to generate vectors using AAV virus.
Herpes simplex virus (HSV) has generated considerable interest in treating nervous system disorders due to its tropism for neuronal cells, but this vector also can be exploited for other tissues given its wide host range. Another factor that makes HSV an attractive vector is the size and organization of the genome. Because HSV is large, incorporation of multiple genes or expression cassettes is less problematic than in other smaller viral systems. In addition, the availability of different viral control sequences with varying performance (temporal, strength, etc.) makes it possible to control expression to a greater extent than in other systems. It also is an advantage that the virus has relatively few spliced messages, further easing genetic manipulations.
HSV also is relatively easy to manipulate and can be grown to high titers. Thus, delivery is less of a problem, both in terms of volumes needed to attain sufficient MOI and in a lessened need for repeat dosings. For a review of HSV as a gene therapy vector, see Glorioso et al. (1995). A person of ordinary skill in the art would be familiar with well-known techniques for use of HSV as vectors.
Vaccinia virus vectors have been used extensively because of the ease of their construction, relatively high levels of expression obtained, wide host range and large capacity for carrying DNA. Vaccinia contains a linear, double-stranded DNA genome of about 186 kb that exhibits a marked “A-T” preference. Inverted terminal repeats of about 10.5 kb flank the genome. The majority of essential genes appear to map within the central region, which is most highly conserved among poxviruses. Estimated open reading frames in vaccinia virus number from 150 to 200. Although both strands are coding, extensive overlap of reading frames is not common.
Other viral vectors may be employed as constructs in the present invention. For example, vectors derived from viruses such as poxvirus may be employed. A molecularly cloned strain of Venezuelan equine encephalitis (VEE) virus has been genetically refined as a replication competent vaccine vector for the expression of heterologous viral proteins (Davis et al., 1996). Studies have demonstrated that VEE infection stimulates potent CTL responses and has been suggested that VEE may be an extremely useful vector for immunizations (Caley et al., 1997). It is contemplated in the present invention, that VEE virus may be useful in targeting dendritic cells.
A polynucleotide may be housed within a viral vector that has been engineered to express a specific binding ligand. The virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell. A novel approach designed to allow specific targeting of retrovirus vectors was developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.
Another approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al., 1989).
2. Nonviral Vectors
Several non-viral methods for the transfer of expression vectors into cells also are contemplated by the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al., 1979) and liofectamine-DNA complex, cell sonication (Fechheimer et al., 1987), gene bombardment using high velocity microprojectiles (Yang et al., 1990), polycations (Bousssif et al., 1995) and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use. A person of ordinary skill in the art would be familiar with the techniques pertaining to use of nonviral vectors, and would understand that other types of nonviral vectors than those disclosed herein are contemplated by the present invention.
In a further embodiment of the invention, the expression cassette may be entrapped in a liposome or lipid formulation. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated is a gene construct complexed with Lipofectamine (Gibco BRL). One of ordinary skill in the art would be familiar with techniques utilizing liposomes and lipid formulations.
Lipid based non-viral formulations provide an alternative to adenoviral gene therapies. Although many cell culture studies have documented lipid based non-viral gene transfer, systemic gene delivery via lipid based formulations has been limited. A major limitation of non-viral lipid based gene delivery is the toxicity of the cationic lipids that comprise the non-viral delivery vehicle. The in vivo toxicity of liposomes partially explains the discrepancy between in vitro and in vivo gene transfer results. Another factor contributing to this contradictory data is the difference in liposome stability in the presence and absence of serum proteins. The interaction between liposomes and serum proteins has a dramatic impact on the stability characteristics of liposomes (Yang and Huang, 1997). Cationic liposomes attract and bind negatively charged serum proteins. Liposomes coated by serum proteins are either dissolved or taken up by macrophages leading to their removal from circulation. Current in vivo liposomal delivery methods use subcutaneous, intradermal, or intracranial injection to avoid the toxicity and stability problems associated with cationic lipids in the circulation. The interaction of liposomes and plasma proteins is responsible for the disparity between the efficiency of in vitro (Felgner et al., 1987) and in vivo gene transfer (Zhu et al., 1993; Solodin et al., 1995; Thierry et al., 1995; Tsukamoto et al., 1995; Aksentijevich et al, 1996).
The production of lipid formulations often is accomplished by sonication or serial extrusion of liposomal mixtures after (I) reverse phase evaporation (II) dehydration-rehydration (III) detergent dialysis and (IV) thin film hydration. Once manufactured, lipid structures can be used to encapsulate compounds that are toxic (chemotherapeutics) or labile (nucleic acids) when in circulation. Liposomal encapsulation has resulted in a lower toxicity and a longer serum half-life for such compounds (Gabizon et al., 1990). Numerous disease treatments are using lipid based gene transfer strategies to enhance conventional or establish novel therapies, in particular therapies for treating hyperproliferative diseases.
G. Screening AssaysThe present invention also contemplates the screening of candidate substances for the ability to modulate growth of a neuron. Particularly preferred candidate substances will be those useful in stimulating directional axonal growth along the A-P axis of the spinal cord. In the screening assays of the present invention, the candidate substance may first be screened for basic biochemical activity and then tested for its ability to modulate activity, at the cellular, tissue or whole animal level. In certain embodiments, an explant assay such as an assay using cultured spinal cord sections may be used in the screening methods. Any method known to those of skill in the art may be used in the claimed invention to conduct the screening assays.
1. Modulators and Assay Formats
a. Assay Formats
The present invention provides methods of screening for modulators of growth of a neuron. In one embodiment, the present invention is directed to a method of:
(a) obtaining a candidate substance;
(b) contacting the candidate substance with a neuron; and
(c) measuring modulation of growth of the neuron.
In an example of yet another embodiment, the assay looks at anterior turning of axons of the neuron.
b. Inhibitors and Activators
An inhibitor according to the present invention may be one which exerts an inhibitory effect on the growth of a neuron. By the same token, an activator according to the present invention may be one which exerts a stimulatory effect on the growth of a neuron.
c. Candidate Substances
As used herein, the term “candidate substance” refers to any molecule that may potentially modulate regeneration of a neuron. The candidate substance may be a protein or fragment thereof, a polypeptide, a peptide, a small molecule inhibitor, or even a nucleic acid molecule. It may prove to be the case that the most useful pharmacological compounds will be compounds that are structurally related to compounds which interact naturally with Wnts, Wnt-like substances, or chemical compounds affecting Wnt signaling pathways. Creating and examining the action of such molecules is known as “rational drug design,” and include making predictions relating to the structure of target molecules.
The goal of rational drug design is to produce structural analogs of biologically active polypeptides or target compounds. By creating such analogs, it is possible to fashion drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules. In one approach, one would generate a three-dimensional structure for a Wnt, and then design a molecule for its ability to interact with the Wnt. Alternatively, one could design a partially functional fragment of a Wnt or a Wnt-like substance (binding, but no activity), thereby creating a competitive inhibitor. This could be accomplished by x-ray crystallography, computer modeling or by a combination of both approaches.
It also is possible to use antibodies to ascertain the structure of a target compound or inhibitor. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of anti-idiotype would be expected to be an analog of the original antigen. The anti-idiotype could then be used to identify and isolate peptides from banks of chemically- or biologically-produced peptides. Selected peptides would then serve as the pharmacore. Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.
On the other hand, one may simply acquire, from various commercial sources, small molecule libraries that are believed to meet the basic criteria for useful drugs in an effort to “brute force” the identification of useful compounds. Screening of such libraries, including combinatorially generated libraries (e.g., peptide libraries), is a rapid and efficient way to screen large number of related (and unrelated) compounds for activity. Combinatorial approaches also lend themselves to rapid evolution of potential drugs by the creation of second, third and fourth generation compounds modeled of active, but otherwise undesirable compounds.
Candidate compounds may include fragments or parts of naturally-occurring compounds or may be found as active combinations of known compounds which are otherwise inactive. It is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds. Thus, it is understood that the candidate substance identified by the present invention may be polypeptide, polynucleotide, small molecule inhibitors or any other compounds that may be designed through rational drug design starting from known modulators of neuronal growth.
Other suitable inhibitors include antisense molecules, ribozymes, and antibodies (including single chain antibodies).
It will, of course, be understood that all the screening methods of the present invention are useful in themselves notwithstanding the fact that effective candidates may not be found. The invention provides methods for screening for such candidates, not solely methods of finding them.
2. In Vitro Assays
A quick, inexpensive and easy assay to run is a binding assay. Binding of a molecule to a target may, in and of itself, be inhibitory, due to steric, allosteric or charge-charge interactions. This can be performed in solution or on a solid phase and can be utilized as a first round screen to rapidly eliminate certain compounds before moving into more sophisticated screening assays. In one embodiment of this kind, the screening of compounds that bind to a Wnt or fragment thereof is provided.
The target may be either free in solution, fixed to a support, expressed in or on the surface of a cell. Either the target or the compound may be labeled, thereby permitting determining of binding. In another embodiment, the assay may measure the inhibition of binding of a target to a natural or artificial substrate or binding partner (such as a Wnt). Competitive binding assays can be performed in which one of the agents (Wnt) is labeled. Usually, the target will be the labeled species, decreasing the chance that the labeling will interfere with the binding moiety's function. One may measure the amount of free label versus bound label to determine binding or inhibition of binding.
A technique for high throughput screening of compounds is described in WO 84/03564. Large numbers of small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with, for example, with a Wnt, and washed. Bound polypeptide is detected by various methods.
Purified target, such as the Wnt, can be coated directly onto plates for use in the aforementioned drug screening techniques. However, non-neutralizing antibodies to the polypeptide can be used to immobilize the polypeptide to a solid phase. Also, fusion proteins containing a reactive region (preferably a terminal region) may be used to link an active region (e.g., the C-terminus of the Wnt) to a solid phase.
Explant culture assays, such as the collagen gel assays described above, are very convenient systems to test the function of the Wnts, Wnt-like substances, and chemical compounds affecting a Wnt signaling pathway in axonal growth and guidance before applying them to animal-based tests. They can also be used as screening methods.
3. In Cyto Assays
Various cell lines that express a Wnt, a Wnt-like substance, and/or a chemical compound affecting a Wnt signaling pathway can be utilized for screening of candidate substances. For example, cells containing a Wnt or a Wnt-like substance with an engineered indicator can be used to study various functional attributes of candidate compounds. In such assays, the compound would be formulated appropriately, given its biochemical nature, and contacted with a target cell.
Depending on the assay, culture may be required. As discussed above, the cell may then be examined by virtue of a number of different physiologic assays (e.g., axon growth). Alternatively, molecular analysis may be performed in which the function of a Wnt or a Wnt-like substance and related pathways may be explored. This involves assays such as those for protein expression, enzyme function, substrate utilization, mRNA expression (including differential display of whole cell or polyA RNA) and others.
4. In Vivo Assays
The present invention particularly contemplates the use of various animal models. Transgenic animals may be created with constructs that permit Wnt expression and activity to be controlled and monitored. The generation of these animals has been described elsewhere in this document.
Treatment of these animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal. Administration will be by any route the could be utilized for clinical or non-clinical purposes, including but not limited to oral, nasal, buccal, or even topical. Alternatively, administration may be by intrathecal, intratracheal instillation, bronchial instillation, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Specifically contemplated are systemic intravenous injection, regional administration via blood or lymph supply.
5. Production of Inhibitors
In an extension of any of the previously described screening assays, the present invention also provide for methods of producing inhibitors. The methods comprising any of the preceding screening steps followed by an additional step of “producing the candidate substance identified as a modulator of” the screened activity.
H. Pharmaceutical PreparationsPharmaceutical preparations of a Wnt, a Wnt-like substance, and/or a chemical compound affecting a Wnt signaling pathway for modulation of growth of a neuron in a mammal are contemplated by the present invention.
1. Formulations
Any type of pharmaceutical preparation of a Wnt, a Wnt-like substance, a chemical compound affecting a Wnt signaling pathway, another axonal guidance molecule, and/or substance that blocks a neuronal inhibitor is contemplated by the current invention. One of skill in art would be familiar with the wide range of types of pharmaceutical preparations that are available, and would be familiar with skills needed to generate these pharmaceutical preparations.
In certain embodiments of the present invention, the pharmaceutical preparation will be an aqueous composition. Aqueous compositions of the present invention comprise an effective amount an of a Wnt, a Wnt-like substance, a chemical compound affecting a Wnt signaling pathway, another axonal guidance molecule, and/or substance that blocks a neuronal inhibitor, and the like, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Aqueous compositions of gene therapy vectors expressing any of the foregoing are also contemplated. The phrases “pharmaceutical composition” or “pharmaceutical preparation” or “pharmacologically effective” or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.
As used herein, “pharmaceutical preparation” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.
The biological material should be extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where appropriate. The active compounds will then generally be formulated for administration by any known route, such as parenteral administration. The preparation of an aqueous composition containing an active agent of the invention disclosed herein as a component or active ingredient will be known to those of skill in the art in light of the present disclosure.
An agent or substance of the present invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. A person of ordinary skill in the art would be familiar with techniques for generation of salt forms. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
The present invention contemplates a Wnt, a Wnt-like substance, a chemical compound affecting a Wnt signaling pathway, another axonal guidance molecule, and/or substance that blocks a neuronal inhibitor that will be in pharmaceutical preparations that are sterile solutions for intravascular injection or for application by any other route. A person of ordinary skill in the art would be familiar with techniques for generating sterile solutions for injection or application by any other route. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients familiar to a person of skill in the art.
Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Formulations for administration via lumbar puncture into the cerebrospinal fluid are also contemplated by the present invention.
The active agents disclosed herein may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.
In addition to the compounds formulated for parenteral administration, such as intravenous injection or via lumbar puncture, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; liposomal formulations; and time release capsules.
Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. A person of ordinary skill in the art would be familiar with well-known techniques for preparation of oral formulations. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 75% of the weight of the unit, or preferably between 25-60%. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.
The use of liposomes and/or nanoparticles is also contemplated for the introduction of the modulator of cell death or gene therapy vectors into host cells. The formation and use of liposomes is generally known to those of skill in the art.
2. Dosage
An effective amount of the therapeutic or preventive agent is determined based on the intended goal, for example inhibition of cell death. The quantity to be administered, both according to number of treatments and dose, depends on the subject to be treated, the state of the subject and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual.
In certain embodiments, it may be desirable to provide a continuous supply of the therapeutic compositions to the patient. For example, following traumatic spinal cord injury, a continuous administration of the therapeutic agent may be administered for a defined period of time, such as direct injection into the cerebrospinal fluid. For various approaches, delayed release formulations could be used that provide limited but constant amounts of the therapeutic agent over an extended period of time. Continuous perfusion of the region of interest may be preferred.
Those of skill in the art are well aware of how to apply gene delivery to in vivo and ex vivo situations. For viral vectors, one generally will prepare a viral vector stock. Depending on the kind of virus and the titer attainable, one will deliver 1×104, 1×105, 1×106, 1×107, 1×108, 1×109, 1×1010, 1×1011 or 1×1012 infectious particles to the patient. Similar figures may be extrapolated for liposomal or other non-viral formulations by comparing relative uptake efficiencies. Formulation as a pharmaceutically acceptable composition is discussed above.
3. Tracers to Monitor Gene Expression Following Administration
Certain embodiments of the present invention employ delivery of a Wnt, a Wnt-like substance, a chemical compound affecting a Wnt signaling pathway, another axonal guidance molecule, and/or substance that blocks a neuronal inhibitor to the target area of interest using expression cassettes. It may be important to determine whether the target site has been effectively contacted with the expression cassette. This may be accomplished by identifying cells in which the expression construct is actively producing the desired polypeptide product. Tagging of the exogenous polypeptide with a tracer element would provide definitive evidence for expression of that molecule and not an endogenous version thereof. Thus, the methods and compositions of the claimed invention may involve tagging of the polypeptide encoded by the expression cassette with a tracer element. A person of ordinary skill in the art would be familiar with these methods of tagging the encoded polypeptide.
I. Combination TherapyIn order to increase the effectiveness of the compositions and methods disclosed herein, it may be desirable to combine a variety of agents into one or more pharmaceutical compositions that can be administered in a regime that is effective in the treatment of the neuronal injuries or disorders described herein. As discussed elsewhere in this specification, those of skill in the art may wish to apply a combination of neuronal attractive, repellant, inhibitory, and/or inhibition blocking substances to the neurons to facilitate appropriate neuronal growth and/or function. This may involve contacting the neuron or spinal cord with these agent(s) at the same time. This may be achieved by contacting the neuron or spinal cord with a single composition or pharmacological formulation that includes multiple agents, or by contacting the cell with two distinct compositions or formulations, at the same time.
Alternatively, the agents may be applied to the neuron or spinal cord in series or succession at intervals ranging from minutes to weeks. In embodiments where two agent are applied separately to the neuron or spinal cord, one may wish ensure that a significant period of time did not expire between the time of each delivery, such that the agents will be able to exert an advantageously combined effect on the neuron(s). In such instances, it is contemplated that one may contact the cell with both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations. In other embodiments, two or more agents applied separately to the neuron or spinal cord with sufficient such that the agents will be able to separately exert their beneficial therapeutic effects on the neurons. In such instances, it is contemplated that one may contact the cell with both modalities In some situations, it may be desirable to extend the time period for treatment such that several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
Various combinations, in an exemplary embodiment, may be employed. For example, any number of regimes may be employed as set forth bells where “A” is a Wnt, Wnt-like substance, or chemical compound effecting a Wnt-signaling pathway and “B” a further Wnt, Wnt-like substance, or chemical compound effecting a Wnt-signaling pathway, a compound providing attractive or repellant guidance to neuronal growth, inhibitor of neuronal growth, or blocker of an inhibitor of neuronal growth:
Administration of the agents to a patient will follow general protocols for the administration as known to those of skill in the art and set-forth herein. It is expected that the treatment cycles may be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the application of the agents.
J. EXAMPLESThe following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1 Materials and MethodsCollagen gel assays. E13 rat spinal cord explants were cultured in collagen gel matrix as described previously (Tessier-Lavigne et al., 1988; Zou et al., 2000). These explants are either “open-book” or post-crossing or pre-crossing for the spinal cord commissural axons. COS7 cells were transfected with various expression constructs with FuGene6 reagent (Roche). The explants were typically cultured for 16-20 hours and fixed in 4% PFA for two hours. The “open-book” explants were analyzed by lipophilic DiI labelling using iontophoresis. The post-crossing explants were stained with a monoclonal antibody (E7) against P3 tubulin (Hybridoma Bank for Developmental Studies). The pre-crossing explants were stained with a monoclonal antibody (4D7) against TAG-1 (Hybridoma Bank for Developmental Studies). Both antibodies were detected using secondary antibodies conjugated with horseradish peroxidase and visualized with 3,3′-diaminobenzene (DAB) (Sigma). Quantification of the post-crossing assays was done as described previously (Zou et al., 2000). The relative total axon bundle length was obtained by normalizing the total length of axons in the presence of Wnt-expressing COS cell aggregates against that in the presence of vector-only transfected COS cell aggregates. The explant assays were performed in three to four sets of multiple explants for each Wnt and an average fold of increase and a standard error were obtained for each Wnt from these sets. Therefore, the relative total length of vector only was defined as 1. n indicates the total number of explants for each construct.
Axon labelling. To reveal the commissural axon projections inside the spinal cord tissue, the inventor used D11 labelling. D11 is a lipophilic dye that becomes highly fluorescent when incorporated in membrane to reveal the shape of the cells and membrane protrusions. In order to focus on relatively smaller numbers of axons and produce more consistent and reproducible injection results, the inventor uses iontophoresis (Fraser, 1996) and point the injection sites with a micromanipulator (Fine Science Tools). DiI was dissolved in MeCl2 (Sigma) at 1 mg/ml. The Dye was delivered into spinal cord tissues with a SD9 current injector (Grass Telefactor). Glass needles were pulled with Narishige PC-10 pipette puller.
In situ hybridization. Mouse E10.9-E13.5 embryos were fixed for either whole-mount or section in situ hybridization as previously described (Keino-Masu et al., 1996; Zou et al., 1997). Specific probes for Wnt1, Wnt4, Wnt6 were obtained by PCR from Wnt1, Wnt4 and Wnt6 constructs in pcDNA1 (Fan et al., 1997) and subcloned into TOPO II vector (Invitrogen). Wnt5a and Wnt7b probes were obtained by RT-PCR from mouse E11.5 embryonic mRNA and subcloned in TOPO II vector.
Immunohistochemistry. E1.5 embryos of frizzled 3 knockout embryos, wild type and heterozygous littermates were fixed for immunohistochemistry with TAG-1 (4D7) antibody as previously described (Serafini et al., 1996).
Wnt and sFRP expression constructs. Wnt1, Wnt4 and Wnt6 full-length cDNA were subcloned into pcDNA3 with Myc epitope tag from pcDNA1 (Fan et al., 1997). Wnt5a expression construct in pCS2 was a kind gift from Dr. Xi He at Children's Hospital at Harvard Medical School and was subcloned into pcDNA3 with Myc epitope tag. Wnt7b cDNA was cloned by RT-PCR from E11.5 mouse embryonic mRNA and subcloned into pcDNA3 with Myc epitope tag. Mouse sFRP1 cDNA construct was a kind gift from Dr. Xi He (Finch et al., 1997). Mouse sFRP2 and sFRP3 cDNAs were cloned by RT-PCR from E1.5 mouse embryonic mRNA and subcloned into pcDNA3 with Myc Epitope tag.
Intrathecal injection. sFRP2 was overexpressed using the bacculovirus system (Lyuksyutova et al., 2003). The overexpressed sFRP2 is tagged with 6×His epitope and can be purified with affinity columns. Purified sFRP2 protein was dialyzed into artificial cerebrospinal fluid and injected into postnatal day 1 mice and rats, followed by one more injection on postnatal day 3. At postnatal day 5, animals were sacrificed, fixed by cardiac perfusion, and dissected for obtaining the spinal cord tissue. Serial sections were obtained along the A-P axis, and the CST axons will be examined by immunohistochemistry.
Behavioral test of injected animals. The functional consequence of sFRP2 injection will be assessed by observing the movement behavior of the injected mice and measuring the strength of the hind paw. A pilot set of experiments with 12 rats and found that 50% of the injected animals displayed a reduction in CST fibers, and that approximately 50% of the injected animals showed splayed hind paws and slowed movement at two weeks after birth.
Example 2 The A-P Guidance Cue(s) is DiffusibleWhen a segment of E13 rat spinal cord is cultured in collagen gel for 16-18 hours, commissural axons were observed to project ventrally, cross the midline and turn anteriorly within the explant, mimicking their in vivo pathfinding. Commissural axon trajectories in these “open-book” explants can be revealed by lipophilic D11 injection into the dorsal side of the explants by iontophoresis (Fraser, 1996). Most of the commissural axons in E13 rat spinal cord “open-book” preparations fixed immediately after dissection (without culturing) are only just approaching the midline or in the process of midline crossing. Therefore, the midline crossing and anterior turning of the commissural axons observed with DiI labeling occurred during the “open-book” culture period.
“Open-book” explants of different anterior-posterior lengths (3 mm, 2 mm, 1 mm and 0.5 mm) were systematically cultured and commissural axon growth was analyzed using focal D11 injection by iontophoresis into the dorsal spinal cord. When the length was reduced to 0.5 mm, abnormal pathfinding behavior of the post-crossing commissural axons was consistently observed, which included knotting, stalling and randomized turning along the A-P axis. This behavior contrasted sharply with that observed in 3 mm explants, in which all axons turned anteriorly. In both short and long explants, commissural axon pathfinding from the dorsal spinal cord to the floor plate was normal. These results were quantified and are shown in
To address whether the A-P guidance cue is attractive or repulsive, DiI was focally injected into the dorsal spinal cord close to the anterior end, in the middle and close to the posterior end of the long explants (3 mm or longer). The axons in the middle and close to the posterior end of the explants were found to always project anteriorly, whereas the axons close to the anterior end almost always make mistakes: they either stall after they cross the midline or they project both anteriorly and posteriorly after midline crossing, or sometimes only posteriorly. The results were quantified using the same criteria as shown in
It is possible that the axons located close to the anterior end of the long explants might be misrouted, because the gradient might be destroyed due to diffusion of the attractant(s) out of the explants, whereas the axons close to the posterior end will turn normally, as the tissue anterior to these turning points will still contain higher concentrations of the attractant(s) (
In order to rule out the possibility that cutting at the anterior end itself produces a repulsive signal, which repels post-crossing commissural axons, studies were conducted to determine whether a cut in the “open-book” explants can prevent axons from projecting rostrally. A cut was introduced within the explants on one side of the “open-book” spinal cords. The spinal cord explants were cultured overnight and the contralateral dorsal spinal cord explants were injected 200 μm-300 μm posterior to the cut site. Commissural axons still projected rostrally and could traverse the cut site, behaving as if they were in the middle of the long “open-book” explants.
Although the cut spinal cords sometimes appeared to be reconnected after overnight culture, they are not sealed back and can be easily separated again at the cut site. And yet, axons can grow through the cut site. This suggests that the A-P gradient of the guidance cue(s) is preserved in such a preparation and a cut (damage) to the spinal cord itself does not produce a cue(s) to repel post-crossing commissural axons. In fact, these axons were faced with two “copies” of cut edge compared to those in short explants. If cut edge produced a repellent, then axons posterior to the internal cut edge would display more severe defects than those in short explants alone. This also demonstrates that the distance between the anterior injection sites and the border of the explants (200 μm-300 μm) is sufficient for commissural axons to turn anteriorly and the failure of anterior turning in short “open-book” explants and at the anterior end of long “open-book” explants is not due to spatial or physical restrictions but rather due to the disruption of the gradient of a guidance cue(s). These results are all consistent with an interpretation that the abnormal axonal behavior at the anterior end of the “open-book” explants is caused by the disruption of a gradient of an attractive molecule(s).
Example 4 Wnt Family Proteins are Candidate A-P Guidance Cue(s)To identify the diffusible guidance cue(s) directing the anterior turn after midline crossing, a candidate gene approach was used. It had been observed that an embryonic limb bud can stimulate the extension of commissural axons only after they have crossed the midline using the “post-crossing” explant assay (Zou et al., 2000). In this assay, commissural axons grow out of the explant after crossing the floor plate, making it possible to test the effects of secreted factors on the axons (see diagram in
If a gradient of diffusible attractant(s) guide commissural axons anteriorly, it might be expected that the tissues anterior to commissural axons can attract post-crossing commissural axons. From previous work of the inventor, both the spinal cord and the floor plate have a potent net repulsive effect to post-crossing commissural axons (Zou et al., 2000). It is possible that the attractant(s) for post-crossing axons are not as diffusible as Semaphorins and Slit proteins precluding the possibility of revealing the function of the attractant(s) in the post-crossing collagen gel assays. Alternatively, the attractant(s) might be expressed in a more restricted fashion and cannot produce a consistently strong attractive effect in assays depending on the orientations of tissues in cultures. In order to circumvent this obstacle and test the model of anterior attractant(s), the function of a major brain target for commissural axons, the ventral-posterior-lateral nucleus of the thalamus, was examined, which is the synaptic target of the spinothalamic tracts (FitzGerald, 1996). The inventor found that the E13.5 ventral-posterior-lateral nucleus can similarly stimulate the extension of the post-crossing commissural axons by three fold (
To determine whether any of these Wnts are likely to affect commissural axon growth in vivo, the expression patterns of Wnts were examined by in situ hybridization in developing mouse embryos during the stages when commissural axons are crossing the midline and turning anteriorly into their longitudinal pathway. Expression of some of these genes in the developing spinal cord has been examined before (Kispert et al., 1996; Liu et al., 2000; Saulnier et al., 2002; Shu et al., 2002; Krylova et al., 2002). At E11.5 (equivalent to E13 rat), Wnt1 is expressed at high levels in the roof plate but diffusely and weakly throughout the spinal cord. Wnt4 is specifically enriched in the floor plate and the ventricular zone and has a decreasing anterior-to-posterior gradient along the entire length of the floor plate at E10.5 as well as E13.5, whereas the expression in the ventricular zone does not show any gradient. A similar anterior-posterior gradient of Wnt4 expression was also observed in the floor plate of E11.5 and E12.5 mouse embryos (data not shown). Wnt5a is expressed widely in the spinal cord but is particularly abundant in the ventral areas of the spinal cord next to the lateral funiculus. Wnt7b is expressed in the ventricular zone of the spinal cord and specifically on the two lateral margins of the floor plate, where the anterior turning of the post-crossing commissural axons occurs. Wnt7b appears to have a decreasing anterior-to-posterior gradient in the ventricular zone but does not display an A-P gradient in the floor plate. Wnt6 and Wnt11 (Kispert et al., 1996) are not expressed in the spinal cord. Wnt3 is expressed in the motor columns but not in the ventral midline or the ventral or lateral funiculi (Krylova et al., 2002) and therefore may not be relevant to commissural axon pathfinding along the anterior-posterior axis. Therefore, several Wnts are expressed in the right place at the right developmental stages to function as regulators of the growth of the post-crossing commissural axons. In particular, the Wnt4 expression displays a clear anterior-posterior gradient along the entire length of the floor plate throughout the time when commissural axons are turning anteriorly after midline crossing (from E10.5 to E13.5). This suggests that Wnt4 might play a role in the anterior-posterior turning decision of post-crossing commissural axons along the entire length of the spinal cord. Interestingly, a similar Wnt4b gradient in the floor plate along the anterior-posterior axis has also been found in zebrafish embryos at similar developmental stages (Liu et al., 2000). Because the ventral posterior lateral nucleus of the thalamus can stimulate the extension of the post-crossing commissural axons, the inventor tested whether any of the Wnt genes are expressed in the thalamus. The inventor found that Wnt1 and Wnt4 genes are expressed at high levels in the thalamus. At E13.5, Wnt4 is expressed in a highly restricted pattern in the thalamus, including the dorsal lateral geniculate nucleus (dLGN) and the ventral-posterior-lateral nucleus (VPL). Wnt1 is also expressed in the dLGN and the VPL at the same stage. Interestingly, Wnt4 and Wnt1 have reciprocal gradients. Wnt4 is expressed at higher level in the dLGN than in the VPL, whereas Wnt1 is expressed at higher level in the VPL than in the dLGN. However, both are expressed in the VPL and the areas used in the explant assays include the VPL. At E11.5, neither Wnt1 nor Wnt4 is expressed in the dorsal diencephalon region destined to be the VPL of the thalamus, consistent with the observation that E11.5 thalamus does not stimulate the extension of the post-crossing commissural axons. Based on the expression pattern of the Wnt genes, the Wnt protein(s) gradient is more likely formed by graded expression levels along the anterior-posterior axis rather than diffusion from the brain targets.
Example 5 SFRPs Can Disrupt Anterior-Posterior Guidance of Commissural AxonsTo test directly whether Wnts are required for the proper anterior turn of the post-crossing commissural axons, potent Wnt inhibitors were used to block the function of all Wnts in the “open-book” explants. Secreted Frizzled-related proteins (sFRPs), are soluble proteins that bind to Wnt proteins with high affinities and thus can block the interaction of Wnts with their receptors, the Frizzleds (Wodarz and Nusse, 1998). sFRPs were produced in the “open-book” collagen gel assays by including sFRP-expressing COS cells in the bottom layer of collagen gel (
It was found that in the presence of any of the three sFRPs (sFRP 1, sFRP2 and sFRP3) or a mixture of all three sFRPs, anterior turning of commissural axons after midline crossing are severely impaired. Instead, they either stall or turn randomly along the anterior-posterior axis, displaying behaviors similar to those observed in the short explant studies discussed above and the anterior injection sites discussed above. In contrast, in the presence of the vector-only-transfected COS cells in the bottom collagen, all commissural axons turned anteriorly after midline crossing. As shown in
In short “open-book” explants, post-crossing axons lose A-P directionality presumably due to the disruption of a Wnt gradient. In order to further test this hypothesis, studies were conducted to determine whether applying a localized anterior source of Wnt protein(s) can rescue the anterior turn of commissural axons after midline crossing in these short explants. The inventor placed COS cell aggregates expressing Wnt4 anterior to the short explants and tested whether the post-crossing axons can turn towards the Wnt4 cell aggregates (
To further test whether Wnt4 can function as an instructive cue to direct axon growth, studies were conducted to determine whether placing COS cell aggregates posterior to the short explants can reorient axons posteriorly (
In order to test whether anterior tissue contain instructive attractant(s) for commissural axons, studies were conducted to attempt to reorient post-crossing commissural axons posteriorly by putting the ventral-posterior thalamus posterior to the “open-book” explants. It was found that in contrast to the Wnt4-overexpressing COS cells, thalamus could not reproducibly reorient axons. The expression of Wnt proteins in the thalamus may not be sufficient to allow Wnt proteins to diffuse into the “open-book” explants to redirect axons. It was found that anterior spinal cord tissue could not reorient axons, either. The spinal cord contains potent repellents to post-crossing commissural axons, such as Sema3B, Sema3F and the Slit proteins, to prevent them from re-entering the grey matter and has a net repulsive effect on post-crossing commissural axons in collagen gel assays (Zou et al., 2000). The Wnt4 protein gradient in the spinal cord is only restricted to the floor plate. The rest of the ventricular zone does not have Wnt4 expression gradient. Therefore, it is very hard to recreate a Wnt4 counter gradient in the “open-book” assay by putting a piece of spinal cord posterior to the explants.
Example 7 Frizzled 3 is Required for Anterior-Posterior Guidance of the Post-Crossing Commissural Axons In VivoThree frizzled genes, which encode receptors for Wnts, fz3, fz8 and fz9, have been found to be expressed in the spinal cord (Borello et al., 1999). This was confirmed by in situ hybridization that fz3, fz8 and fz9 are indeed expressed in the spinal cord from E9.5 to E13.5 during the time when commissural axons are making anterior turns. Among the three frizzleds, fz3 is the most relevant, because it is expressed broadly in the spinal cord, covering the area where commissural neuron cell bodies are located. Interestingly, fz3 transcripts appear to be enriched in the ventral funiculi where post-crossing commissural axons are located at a E11.5, when a large number of commissural axons have already crossed the midline. Fz8 is expressed more weakly and is not expressed in the most dorsal portion of the spinal cord. Fz9 is only expressed in the ventricular zone where non-differentiated neurons are localized but not in the dorsal mantle zone where commissural neuron cell bodies are located. Commissural axon projections in fz3 knockout embryos (Wang et al., 2002) were examined by immunohistochemistry and DiI labeling with a monoclonal antibody against TAG-1, a commissural axonal marker that only labels the pre-crossing and the midline crossing segments of the commissural axons but not the post-crossing segment of the commissural axons. It was found that the dorsal-ventral projection of pre-crossing commissural axons were normal compared to wild type control, but post-crossing commissural axons projected randomly along the anterior-posterior axis after midline crossing with 100% penetrance. From crosses between fz3 heterozygotes, four litters among which were seven homozygous mutants were examined. For three of these litters, the dissected spinal cords were analyzed without knowledge of their genotypes. In these blinded experiments, 5/5 mutant and 11/11 wild type or heterozygous spinal cords were correctly identified; the probability of this occurring by chance is 4×10−5. It was found that in all injection sites, commissural axons either turned randomly along the anterior-posterior axis or stalled after midline crossing, whereas their pre-crossing trajectory was normal, consistent with the observations discussed above using explant assays, suggesting that the Wnt/Frizzled pathway is only required for anterior-posterior axon guidance after midline crossing in vivo. As previously reported, no spinal cord patterning defects were observed in the fz3 knockout mice at this stage of development as assessed by markers such as Nkx2.2, HNF-3β, Lim2, and Isl1 (Wang et al., 2002). Both the dorsal-ventral and anterior-posterior pathfinding of commissural axons are normal in LRP6−/− embryos although dramatic patterning defects were observed in these animals (Pinson et al., 2000), suggesting that the canonical Wnt/β-catenin signaling pathway is not involved in the differentiation, the dorsal-ventral pathfinding and the anterior-posterior guidance decision of commissural axons at the midline.
Example 8 Wnt Genes are Expressed in a “Half-Pipe” Gradient Along the Neonatal Spinal CordBecause corticospinal tract axons project posteriorly along the dorsal funiculus of the spinal cord, the inventor examined the expression pattern of Wnt genes around the dorsal funiculus by in situ hybridization. The inventor cloned the entire family of rodent Wnt genes (including 19 members) and performed in situ hybridization at postnatal days 0 and 3 along the anterior-posterior axis. The inventor found that five Wnt genes are expressed in the dorsal midline and dorsal funiculus. Wnt1, and Wnt5a are expressed at a higher level. The other Wnts, Wnt7b, Wnt8a, and Wnt9a, are expressed at lower levels. Along the anterior-posterior axis, all of these Wnt genes have a high-to-low gradient from the cervical and thoracic level. Intriguingly, all these Wnt genes display a reverse gradient at the lumbar level: low-to-high gradient. Therefore, multiple Wnt genes are expressed in a biphasic gradient, or “half-pipe” gradient.
The biphasic gradient along the entire spinal cord suggests that Wnts first “push” CST axons posteriorly along the cervical and thoracic cord but then act as stop signal to terminate the CST axons at the lumber cord, much like the motion in a “half-pipe”.
Example 9 Wnt Proteins Repel Frontal Cortical AxonsIn order to test whether Wnts can guide corticospinal tract axons, the inventor performed explant assays to evaluate the function of Wnt proteins in frontal cortical axons in collagen gel. Postnatal day 0 brains were dissected out and sliced with tissue chopper. Layer 5 cortical explants were dissected from the frontal motor cortical region and culture in collagen for 60 hours. Long axons grew out in the collagen gel and are stained positively with a corticospinal tract marker, a monoclonal antibody against N-CAM, 5A5. COS cells were transfected with Wnt expression constructs and made into cell aggregates, and the inventor positioned the cell aggregates next to the cortical explants dissected out from postnatal P0 frontal cortex. The inventor found that Wnt1 protein potently inhibits the outgrowth of axons from the frontal cortex in these assays, suggesting that corticospinal tract axons might respond to Wnt proteins as they pathfind along the spinal cord in vivo. Very few axons grew out in the collagen gel, and the axon's length is much reduced as well. A slight repulsive effect can be observed. To address the possibility that the cell aggregates may be secreting too much Wnt1 protein so that axons cannot grow out of the explants, the inventor diluted the transfected COS cells with untransfected COS cells and found that Wnt1 shows robust repulsion when diluted. The inventor tested the function of Wnt1 on E18.5 cortical axons and found Wnt1 can only weakly repel frontal cortical axons. CST axons reach the spinal cord at P0. At E18.5, the CST axons are still in the midbrain and the hindbrain. The time course of Wnt1 responsiveness is consistent with it role in CST axon pathfinding once CST axons enter the spinal cord. Wnt5a also repel postnatal motor cortical axons.
Example 10 Wnt Proteins Also Regulate the A-P Pathfinding of the CST AxonsThe inventor found that several Wnt genes are expressed in a high-to-low gradient in the gray matter cupping the dorsal funiculus from the cervical to the thoracic spinal cord where corticospinal tract axons first enter the spinal cord and project posteriorly at postnatal day 0. At the lumbar spinal cord, Wnt gene expression in the gray matter displays a reversed gradient (low-to-high) forming a “half-pipe” gradient along the entire length of the spinal cord. Such gradient persists from P0 to at least P5. The functional studies showed that Wnt proteins could repel axons from frontal motor cortex in a collagen gel assay. Therefore, first gradient guides CST axons to project from the cervical cord to the thoracic cord, and the second reverse gradient helps to stop CST axons at the lumbar level.
Example 11 A Repulsive Wnt Receptor, Ryk, is Expressed in the CST AxonsAlong the Entire A-P trajectory Axon guidance molecules are often bi-functional, attracting some axons while repelling others, depending on the guidance receptor composition in the responding neurons. Vertebrate commissural axons are attracted by Wnts, whereas frontal cortical axons are repelled by Wnts. In Drosophila, Wnt5 was found to play a repulsive role in the pathway selection before midline crossing (Yoshikawa et al., 2003). This repulsion is mediated by a Wnt receptor called Derailed through direct binding and is independent of Frizzled (Yoshikawa et al., 2003). The inventor found that the vertebrate Derailed, Ryk (Halford et al., 2000), is not expressed in commissural axons, although Frizzled3 is, and Frizzled3 is required for mediating Wnt attraction (Lyuksyutova et al., 2003).
Further investigating why the cortical axons are repelled by Wnts, the inventor first generated an in situ probe for Ryk and found that the Ryk gene is expressed in layers 5 and 6 of the frontal cortex. The levels of Ryk expression at E18.5 are much lower than are that of P0. The inventor obtained a published antibody against the mouse Ryk protein (Kamitori et al., 2002) and performed immunohistochemistry, and the inventor found that Ryk protein is present in layer 5 neurons and is present in the internal capsule of E18.5 brain. The inventor then generated polyclonal antibodies against the extracellular domain of Ryk and further confirmed that Ryk protein is present in the CST axons forming the pyramidal decussation and the pyramidal tracts in the dorsal funiculus of the spinal cord. Therefore, Ryk is expressed in the CST axons at the right time to mediates Wnt repulsion.
Example 12 Ryk Antibodies can Block the Repulsion of CST Axons by WntsTo demonstrate that Ryk is involved in mediating Wnt repulsion in vertebrate axons, the inventor used the polyclonal antibodies generated against the ectodomain of Ryk and tested whether the Ryk antibodies can block the repulsion by Wnts in collagen gel assays. The inventor found that addition of purified Ryk antibodies in collagen gel assays blocked the repulsive effects of Wnt proteins, suggesting that Ryk does mediate Wnt repulsion in vertebrates and may play important roles in CST axon guidance such as the anterior-posterior guidance of CST axons in vivo. The inventor found that in the presence of Wnt1 protein, frontal cortical axons tend to grow much shorter and away from the pointed source of Wnt1. When Ryk antibodies were included, frontal cortical axons were no longer repelled, and the outgrowth was increased.
Example 13 Intrathecal Injection of sFRP2 Protein at Cervical Level Caused Reduction of CST Fibers in the Dorsal Funiculus and Impaired Motor FunctionTo address the in vivo function of the repulsive effects of Wnt proteins on corticospinal tract axon guidance, the inventor injected purified sFRP2 protein to postnatal cervical spinal cord at P1, P3 and then analyzed the CST axon projection in P5 spinal cord. Transverse section of the vehicle and sFRP2 injected animals were collected every 800 um along the entire A-P axis of the P5 spinal cord and stained with a CST marker 5A5. The inventor found that the dorsal funiculus areas are much reduced in injected animals, suggesting that the posterior growth of CST axons was interfered. Similar results were obtained from multiple groups of mice and rats. Some animals were raised to adulthood and their motor functions were analyzed. The inventor found that the sFRP2 injected animals display consistent weakening of grip strength throughout the entire period of the tests, suggesting the posterior growth defects caused by sFPR2 injection interfered motor system development.
These studies suggest that Wnt proteins control not only the guidance of ascending sensory axons, but also that of the descending motor pathways through a Ryk-dependent signaling pathway.
Example 14 Additional Studies Involving Injection of Wnt Inhibitors into Spinal CordsIn addition to the studies described above, the sFRP2 protein has also been injected to the lumbar and sacral spinal cord on postnatal day 5 and 7 and animals were fixed on day 9. Data obtained from these studies will indicate whether inhibiting Wnt function in the posterior portion of the spinal cord will cause overshooting of corticospinal tract axons, leading to abnormal development of the motor system, and provide further information allowing one of skill to develop appropriate regimes for spinal cord regeneration.
Additionally, Anti-Ryk antibodies have also been injected to both the cervical and lumbar spinal cord regions to allow for the analysis of anatomical defects of motor axon growth and behavioral defects. These studies, have confirmed that Ryk is an inhibitor of Wnt-mediated action on neurons and a target for therapeutics.
Example 15 In Situ Hybridization Studies of Wnts Expression in Normal and Injured Spinal CordsTo study patterns of Wnt expression, the inventor cloned the entire family of Wnts and performed in situ hybridization. Most of the Wnts are no longer expressed in the adult spinal cord. One Wnt gene, Wnt5a, is expressed highly in the spinal cord. Wnt8a is weakly expressed.
Researchers have found that it is possible to regenerate sensory axons by blocking inhibitors of axon growth but it is nearly impossible to regenerate corticospinal cord (Sivasankaran et al. 2004). It is possible to that the Wnt5a is expressed in the adult spinal cord and other Wnts that become induced at injured sites in the spinal cord result in inhibition of normal cord growth. Because corticospinal tract axons are repelled by Wnts and sensory axons are attracted by Wnts, abnormal Wnt production after injury can result in selective inhibition of the motor cortical axons in the spinal cord. Any injury-induced Wnts, together with Wnt5a, may cause a repulsive environment so that the adult axons fail to regenerate.
One can use the data from in situ studies of normal and injured spinal cords to study whether various Wnt genes are induced upon spinal cord injury. To obtain data from injured spinal cords, an adult mouse spinal cord can be lesioned at cervical and thoracic levels by a hemi-section injury paradigm. The animals can be fixed at day 1, 7, 14 and one month after injury and the expression patterns of Wnt genes determined by in situ hybridization and compared to data from uninjured spinal cords.
Data from the studies described in this example can be used to determine appropriate substances to use to prevent any injury-induced Wnts from preventing proper neuronal regeneration.
Example 16 Transgenic Mice StudiesIn order to further demonstrate the roles of Wnts in neuronal guidance and regeneration, a variety of transgenic mice lines were created. In these lines, generally, a dominant-negative inhibitor transgene is expressed to produce an inhibitor of a Wnt inside relevant neurons. These transgenic mice are produced by methods well-known to those of skill in the art.
For example, transgenic mice lines expressing specific dominant-negative inhibitors of Wnt intracellular signaling (dominant-negative disheveled) in a subset (Neurogenin-2 expressing) of commissural neurons (which likely give rise to pain sensory pathway and are attracted by Wnts to project to the brain) showed a kangaroo gait phenotype in the hindlimb. The gait (hopping) behavior appears to depend on the texture of the surface the mice were walking on, suggesting sensory system defects in these neuron. These data demonstrate that Wnt signaling is important for the normal wiring of the nervous system and support a cell-autonomous mechanism, meaning that the Wnt signaling pathway is required in the neurons which are responding to the Wnt gradient.
One can conduct further transgenic animal studies to show the roles in which the Wnt signaling pathway in axon sensory axon guidance and provide tools for axonal regeneration inhibitors of Wnt signaling. In this regard, dominant negative transgenic animals can be created in subsets of commissural neurons to further test the role of Wnt signaling pathway in commissural neurons. Axonal projection and mouse behavior can be analyzed in these animals.
Further once transgenic mice are created, spinal cord lesion experiments as described above can be carried out and the Wnt expression pattern in the injured spinal cord analyzed. The function of Wnts in adult spinal cord axons can be tested to see whether Wnts continue to attract sensory axons and repel motor axons. If Wnt attract sensory axons, the induction of Wnts may be helpful for axon regeneration. But if Wnts repel motor axons, the induced Wnts in the spinal cord will block regenerative growth of motor axons. In this case, anti-Ryk antibody, which blocks the repulsive function of Wnt specifically will be applied to block the inhibitory effects of Wnts on motor axon regeneration. Alternatively, interference of the Ryk signaling specifically will also block the repulsion, allowing regeneration to occur. These results will provide insights to how Wnts can be used to help spinal cord axon regeneration.
Example 17 Psychoactive Drugs in Combination with Wnt TherapyPsychoactive drugs, such as amphetamine, improve functional recovery following stroke in experimental animals, suggesting a role in promoting nerve repair and regeneration (Long and Young, 2003). In view of the teachings of this specification, those of skill will be able to determine the effects of these drugs on Wnt signaling, axon guidance, and regeneration. Those of skill will then be able to further modify such drugs and/or their treatment regimes to enhance the drug effect on regeneration and reduce side effects without losing the effect on regeneration.
It is expected, in view of the teachings of this specification, that a combination of Wnt inhibitors or psychoactive drugs will be beneficial in promoting axonal regeneration.
Example 18 Wnts Pattern Synaptic ConnectionsWnts not only are axon guidance molecules controlling pathfinding of axons toward their targets but also play important roles in patterning the synaptic connections once they reach their target. This process of target selection ensures the specific neuron to neuron connection and is essential to the development of the functional circuits throughout the nervous system. The inventor has found, at least in the somatosensory system and the visual system, Wnts play critical roles in patterning these synaptic target connections to establish topographic map. For example, when, in an animal model, Wnts are mis-expressed in the synaptic neuronal target area, the tectum, there is misconnection of the axons at the tectum and a resulting disrupted target map, causing the animals to be blind. Likewise, if Ryk is inhibited in a transgenic mouse in which a dominant-negative Ryk inhibitor is expressed in retinal ganglia cell neurons, similar results occur.
These studies suggest that Wnts play a role in patterning synapic connections and that the Wnt pathway can be modulated in manners discussed elsewhere in this specification to ensure specific synaptic reconnection in repair damaged neural circuits.
Example 19 Testing of Wnt, Wnt-Like Substances, and Compounds Affecting a Wnt Signaling PathwayBased on the disclosure of the specification and the knowledge available to one of ordinary skill in the art, Wnts, Wnt-like substances, chemical compounds affecting a Wnt signaling pathway, sFRPs, sFRP-like substances, Ryk, Ryk-like substances, blockers of neuronal growth inhibitors, neuronal growth inhibitors, and/or repulsive and attractive neuronal guidance molecules can be identified. The candidate substances that have been identified can then be tested in accordance with the techniques disclosed in the specification, and evaluated for the ability to modulate neuronal growth. Testing can be conducted in vitro, such as by use of the previously disclosed explant assay, or in vivo in animal models of neuronal damage. One of ordinary skill in the art would be familiar with the numerous methods and techniques that can be employed to test candidate substances affecting a Wnt signaling pathway for ability to promote neuronal growth and regeneration.
Example 20 Clinical Trials of the Use of a Wnts, Wnt-Like Substances, and/or Chemical Compounds Affecting a Wnt Signaling Pathway in the Treatment of Diseases in GeneralThis example is generally concerned with the development of human treatment protocols using Wnts, Wnt-like substances, chemical compounds affecting a Wnt signaling pathway, sFRPs, sFRP-like substances, Ryk, Ryk-like substances, blockers of neuronal growth inhibitors, neuronal growth inhibitors, and/or repulsive and attractive neuronal guidance molecules in the treatment of diseases such as those previously discussed in this specification. In particular, such drug treatment can be of use in the clinical treatment of various diseases in which neuronal dysfunction plays a role. Examples of these diseases include traumatic spinal cord injury. A more detailed example pertaining to traumatic spinal cord injury is discussed in the next example.
The various elements of conducting a clinical trial, including patient treatment and monitoring, will be known to those of skill in the art in light of the present disclosure. The following information can be used as a general guideline for use in establishing use of Wnts, Wnt-like substances, chemical compounds affecting a Wnt signaling pathway, sFRPs, sFRP-like substances, Ryk, Ryk-like substances, blockers of neuronal growth inhibitors, neuronal growth inhibitors, and/or repulsive and attractive neuronal guidance molecules in clinical trials. Patients with the targeted disease can be newly diagnosed patients or patients with existing disease. Patients with existing disease may include those who have failed to respond to at least one course of conventional therapy.
The Wnts, Wnt-like substances, chemical compounds affecting a Wnt signaling pathway, sFRPs, sFRP-like substances, Ryk, Ryk-like substances, blockers of neuronal growth inhibitors, neuronal growth inhibitors, and/or repulsive and attractive neuronal guidance molecules may be administered alone or in combination with the another therapeutic agent. The agents may be administered intravenously, directly into the cerebrospinal fluid, or by another mechanism that is specific to the disease that is being treated. The agent may also be administered intraoperatively, such as by direct application to the spinal cord during surgery.
The starting dose may, for example, be 0.5 mg/kg body weight. Three patients may be treated at each dose level in the absence of a defined level of toxicity. Dose escalation may be done by 100% increments (e.g., 0.5 mg, 1 mg, 2 mg, 4 mg) until drug related toxicity of a specific level develops. Thereafter dose escalation may proceed by 25% increments. The administered dose may be fractionated.
The therapeutic agent may be administered over a short infusion time or at a steady rate of infusion over a period of days. The infusion may be administered alone or in combination with other agents. The infusion given at any dose level will be dependent upon the toxicity achieved after each.
Physical examination, laboratory tests, and other clinical studies specific to the disease being treated may, of course, be performed before treatment and at intervals of about 3-4 weeks later. Laboratory studies can include CBC, differential and platelet count, urinalysis, SMA-12-100 (liver and renal function tests), coagulation profile, and any other appropriate chemistry studies to determine the extent of disease, or determine the cause of existing symptoms. If necessary, appropriate biological markers in serum can be monitored.
Example 21 Clinical Trials of the Use of a Wnt or a Wnt-Like Substance or Chemical Compounds affecting a Wnt Signaling Pathways in the Treatment of Spinal Cord InjuryThis example is concerned with the development of human treatment protocols using a Wnts, Wnt-like substances, chemical compounds affecting a Wnt signaling pathway, sFRPs, sFRP-like substances, Ryk, Ryk-like substances, blockers of neuronal growth inhibitors, neuronal growth inhibitors, and/or repulsive and attractive neuronal guidance molecules in the treatment of spinal cord injury. The various elements of conducting a clinical trial, including patient treatment and monitoring, will be known to those of skill in the art in light of the present disclosure. The following information can be used as a general guideline for use in establishing clinical trials pertaining to spinal cord treatment.
Patients with spinal cord injury for clinical study will typically have failed to respond to at least one course of conventional therapy. Measurable disease is not required.
The therapeutic agent may be administered alone or in combination with the another chemotherapeutic agent. The administration may be intravenously, directly into or around the spinal cord, or in any other manner known to those of skill in the art. The starting dose may be 0.5 mg/kg body weight. Three patients may be treated at each dose level in the absence of grade >3 toxicity. Dose escalation may be done by 100% increments (0.5 mg, 1 mg, 2 mg, 4 mg) until toxicity is detected. Thereafter dose escalation may proceed by 25% increments.
The therapeutic agent may be administered over a short infusion time or at a steady rate of infusion over a 7 to 21 day period. The agent may be administered alone or in combination with agents for treatment of spinal cord injury. The infusion given at any dose level will be dependent upon the toxicity achieved after each. Increasing doses of the Wnts, Wnt-like substances, chemical compounds affecting a Wnt signaling pathway, sFRPs, sFRP-like substances, Ryk, Ryk-like substances, blockers of neuronal growth inhibitors, neuronal growth inhibitors, and/or repulsive and attractive neuronal guidance molecules, in combination with other therapeutic agents will be administered to groups of patients until approximately 60% of patients show unacceptable toxicity. Doses that are 2/3 of this value could be defined as the safe dose.
Physical examination, neurological function, and laboratory tests can, of course, be performed before treatment and at intervals of about 3-4 weeks later. Laboratory studies should include CBC, differential and platelet count, urinalysis, SMA-12-100 (liver and renal function tests), coagulation profile, and any other appropriate chemistry studies to determine the extent of disease, or determine the cause of existing symptoms. Also appropriate biological markers in serum can be monitored.
To monitor disease course and evaluate the response, it is contemplated that the patients may be examined for neurological function. Laboratory studies such as a CBC, differential and platelet count, coagulation profile, and/or SMA-12-100 shall be performed weekly. Appropriate clinical studies such as radiological studies should be performed and repeated every 8 weeks to evaluate response.
Clinical response may be defined by acceptable measure. For example, a response may be defined by improvement in neurological dysfunction, and can be graded using parameters known to those of skill in the art.
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
REFERENCESThe following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
- U.S. Pat. No. 4,554,101
- U.S. Pat. No. 4,797,368
- U.S. Pat. No. 5,139,941
- Aksentijevich et al., Hum. Gene Ther., 7(9):1111-1122, 1996.
- Altman et al., Adv. Anat. Embryol Cell Biol., 85:1-164, 1984.
- Augsburger et al., Neuron, 24:127-141, 1999.
- Borello et al., Mech. Dev., 89:173-177, 1999.
- Boussif et al., Proc. Natl. Acad. Sci. USA, 92(16):7297-7301, 1995.
- Bradley and Brown, EMBO J., 9:1569-1575, 1990.
- Bueno et al., Int. J. Dev. Biol. Suppl. 1:79 S-80S, 1996.
- Caley et al., J. Virology, 71(4):3031-3038, 1997.
- Carbonelli et al., FEMS Microbiol. Lett., 177(1):75-82, 1999.
- Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987.
- Christiansen et al., Mech. Dev., 51:341-350, 1995.
- Cocea, Biotechniques, 23(5):814-816, 1997.
- Coffin, In: Virology, Fields et al., eds., Raven Press, NY, 1437-1500, 1990.
- Couch et al., Am. Rev. Resp. Dis., 88:394-403, 1963.
- Davis et al., Curr. Biol., 6:146-148, 1996.
- Dealy et al., Mech. Dev., 43:175-186, 1993.
- Derossi et al., J. Biol. Chem., 269:10444-10450, 1994.
- Dickson, Science, 298:1959-1964, 2002.
- Ebens et al., Neuron, 17:1157-1172, 1996.
- Elliott and O'Hare, Cell, 88:223-233, 1997.
- Fan et al., Dev. Biol., 191:160-165, 1997.
- Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84:8463-8467, 1987.
- Finch et al., Proc. Natl. Acad. Sci. USA, 94:6770-6775, 1997.
- FitzGerald, In: Neuroanatomy Basic and Clinical, W. B. Sauders Company LTD, 1996.
- Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348-3352, 1979.
- Francis et al., Development, 120:209-218, 1994.
- Fraser, In: Methods in Cell Biology, 147-160, Academic Press, Inc., 1996.
- Gabizon et al., Cancer Res., 50(19):6371-6378, 1990.
- Gavin et al., Genes Dev., 4:2319-2332, 1990.
- Glorioso et al., Mol. Biotechnol., 4(1):87-99, 1995.
- Gopal, Mol. Cell Biol., 5:1188-1190, 1985.
- Graham and Van Der Eb, Virology, 52:456-467, 1973.
- Grunhaus et al., Seminar in Virology, 200(2):535-546, 1992.
- Halford et al., Nat. Genet., 25:414-418, 2000.
- Hall et al., Cell, 100:525-535, 2000.
- Harland and Weintraub, J. Cell Biol., 101: 1094-1099, 1985.
- Hofmann et al., Dev. Genet., 19:43-50, 1996.
- Joosten et al., Brain Res. Dev. Brain Res., 94:99-105, 1996.
- Kamitori et al., Brain Res. Mol. Brain Res., 104:255-266, 2002.
- Keino-Masu et al., Cell, 87:175-185, 1996.
- Kennedy et al., Cell, 78:425-435, 1994.
- Kispert et al., Development, 122:3627-3637, 1996.
- Klein and Melton, Proc. Natl. Acad. Sci. USA, 93:8455-59, 1996.
- Klingensmith and Nusse, Dev. Biol., 166:396-414, 1994.
- Kotin et al., Proc. Natl. Acad. Sci. USA, 87(6):2211-5, 1990.
- Krylova et al., Neuron, 35:1043-1056, 2002.
- Laughlin et al., J. Virol., 60(2):515-524, 1986.
- Lebkowski et al., Mol. Cell. Biol., 8(10):3988-3996, 1988.
- Levenson et al., Hum Gene Ther, 9(8):1233-6, 1998.
- Liu et al., Mech. Dev., 91:409-413, 2000.
- Long and Young, Qjm 96:673-685, 2003.
- Lucas and Salinas, Dev. Bio., 192:31-44, 1997.
- Lyuksyutova et al., Science, 302:1984-1988, 2003.
- McCarty et al., J. Virol., 65(6):2936-2945, 1991.
- McLaughlin et al., J. Virol., 62(6):1963-1973, 1988.
- McMahon, Trends Genet., 8:236-242, 1992.
- McMahon and Bradley, Cell, 62:1073-1085, 1990.
- Miller, Genome Biology, 3(1):3001.1-3001.15, 2001.
- Morata and Lawrence, Dev. Biol., 56:227-240, 1977.
- Muzyczka, Curr Top Microbiol Immunol, 158:97-129, 1992.
- Nagahara et al., Nature Medicine, 4:1449-1452, 1998.
- Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982.
- Nusse and Varmus, Cell, 69:1073-1087, 1992.
- Papkoff and Schryver, Mol. Cell Biol., 10:2723-2730, 1990.
- Parr et al., Dev. Biol., 237:324-332, 2001.
- Paxinos, In: The Rat Nervous System, 2nd Ed., Academic Press, 1995.
- Pelletier and Sonenberg, Nature, 334:320-325, 1988.
- Pinson et al., Nature, 407:535-538, 2000.
- Potter et al., Proc. Natl. Acad. Sci. USA, 81:7161-7165, 1984.
- Ramon y Cajal, La Cellule, 9:119-258, 1893.
- Rijsenijk et al., Cell, 50:649-657, 1987.
- Rippe et al., Mol. Cell Biol., 10:689-695, 1990.
- Roux et al., Proc. Nat'l Acad. Sci. USA, 86:9079-9083, 1989.
- Samulski et al., EMBO J., 10:3941-3950, 1991.
- Samulski et al., J. Virol, 63:3822-3828, 1989.
- Saulnier et al., Dev. Biol., 248:13-28, 2002.
- Serafini et al., Cell, 78:409-424, 1994.
- Serafini et al., Cell, 87:1001-1014, 1996.
- Shelling and Smith, Gene Therapy, 1: 165-169, 1994.
- Shirasaki et al., Science, 279:105-107, 1998.
- Shu et al., Development, 129:4831-4842, 2002.
- Sivasankaran et al., Nat Neurosci 7:261-268, 2004.
- Solodin et al., Biochemistry, 34(41):13537-13544, 1995.
- Tessier-Lavigne et al., Nature, 336:775-758, 1988.
- Tessier-Lavigne et al., Science, 274:1123-1133, 1996.
- Tessier-Lavigne, Curr. Opin. Genet. Dev., 4:596-601, 1994.
- Thierry et al., Proc. Natl. Acad. Sci. USA, 92(21):9742-9746, 1995.
- Thomas and Cappechi, Nature, 346:847-850, 1990.
- Top et al., J. Infect. Dis., 124:155-160, 1971.
- Tratschin et al., Mol. Cell Biol., 4:2072-2081, 1984.
- Tsukamoto et al., Nat. Genet., 9(3):243-248, 1995.
- Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986.
- Ungar et al., Mech. Dev., 52:153-164, 1995.
- Vant Veer et al., Mol. Cell Biol., 4:2532-2534, 1984.
- Wagner et al., Science, 260:1510-1513, 1993.
- Wang et al., J. Neurosci., 22:8563-8573, 2002.
- Wodarz et al., Annu. Rev. Cell Dev. Biol., 14:59-88, 1998.
- Wu and Wu, Biochemistry, 27:887-892, 1988.
- Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987.
- Yang et al., Proc Natl. Acad Sci. USA, 87:9568-9572, 1990.
- Yoshikawa et al., Nature, 422(6932):583-588, 2003.
- Zakany et al., Nature, 362:546-549, 1993.
- Zhu et al., Science, 261(5118):209-211, 1993.
- Zou et al., Cell, 102:363-375, 2000.
- Zou, et al., Development, 124:793-804, 1997.
Claims
1. A method for modulating the directional growth of a mammalian neuron comprising contacting the neuron with an inhibitor of frizzled.
2. The method of claim 1, wherein the frizzled inhibitor comprises a frizzled antibody.
3. The method of claim 2, wherein the frizzled antibody inhibits a frizzled selected from the group consisting of frizzled3, frizzled8, and frizzled9.
4. The method of claim 1, wherein the neuron is contacted with the inhibitor in a spinal cord.
5. The method of claim 4, wherein the inhibitor is provided as a concentration gradient.
6. The method of claim 5, wherein the concentration gradient is provided as a decreasing anterior-posterior concentration gradient along the spinal cord.
7. The method of claim 4, wherein the directional growth of the neuron occurs along the anterior-posterior axis of the spinal cord.
8. The method of claim 4, wherein the directional growth of the neuron is along the spinothalamic pathway.
9. The method of claim 4, wherein the spinal cord has been damaged.
10. The method of claim 1, wherein the neuron is a commissural neuron.
11. The method of claim 10, wherein the commissural neuron is contacted with the inhibitor post-midline crossing.
12. The method of claim 1, wherein the frizzled inhibitor comprises a sFRP.
13. The method of claim 12, wherein the sFRP is selected from sFRP1, sFRP2 and sFRP3.
14. The method of claim 1, wherein the neuron is further contacted with a neuronal growth inhibitor.
15. The method of claim 1, wherein the neuron is further contacted with a substance that blocks activity of a neuronal growth inhibitor.
16. The method of claim 1, wherein the neuron is a motor neuron or a sensory neuron.
17. The method of claim 1, wherein the neuron is a damaged neuron.
18. The method of claim 17, wherein the directional growth of the neuron facilitates regeneration of the neuron.
19. The method of claim 1, wherein the inhibitor repels neuronal growth.
20. The method of claim 1, wherein the inhibitor is provided as a pharmaceutical composition.
Type: Application
Filed: May 20, 2008
Publication Date: Dec 4, 2008
Applicant: THE UNIVERSITY OF CHICAGO (Chicago, IL)
Inventor: Yimin Zou (Chicago, IL)
Application Number: 12/123,721
International Classification: A61K 38/17 (20060101); C12N 5/06 (20060101); A61K 39/395 (20060101); A61P 25/00 (20060101);
