Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-12-11
2004-03-23
Falk, Anne-Marie (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S004000, C435S006120, C435S375000, C435S440000, C435S456000, C435S476000
Reexamination Certificate
active
06709866
ABSTRACT:
The field of the invention is neuronal cell death.
BACKGROUND OF THE INVENTION
Neuronal cell death can occur as a result of a variety of conditions including traumatic injury, ischemia, degenerative disease (e.g., Parkinson's disease, ALS, or SMA), or as a normal part of tissue development and maintenance.
We have previously discovered the NAIP and the IAP proteins (see U.S. Ser. No. 08/511,485 now U.S. Pat. No. 5,919,912; Ser. No. 08/576,956 now U.S. Pat. No. 6,156,535; and 60/017,354, incorporated by reference). These proteins are involved in the control of apoptosis.
Developmental cell death, or apoptosis, is a naturally occurring process thought to play a critical role in establishing appropriate neuronal connections in the developing central nervous system (CNS). Apoptosis is characterized morphologically by condensation of the chromatin followed by shrinkage of the cell body. Biochemically, the hallmark of apoptosis is the degradation of nuclear DNA into oligonucleosomal fragments (multiples of 180 basepairs) mediated by a Ca
2+
/Mg
2+
-dependent endonuclease. DNA laddering precedes cell death and may be a key event leading to death. In keeping with this proposal, agents which inhibit DNA fragmentation prevent apoptosis, whereas morphology indicative of apoptosis is produced by enzymes that digest nuclear DNA. Apoptosis is often dependent on RNA and protein synthesis within the dying cell suggesting the activation of a cell death pathway. The best defined genetic pathway of cell death is in the nematode Caenorhabditis where both effector (ced-3 and ced-4) and repressor (ced-9) genes have been isolated. Similar genes have been identified in mammals. One such example is the proto-oncogene bcl-2 which is thought to be the mammalian homolog of ced-9. Overexpression of Bcl-2 has been shown to render neurons resistant to the damaging effects of a wide variety of noxious treatments. However, the very low levels of Bcl-2 detected in adult brain suggest that other proteins may play a more important role in preventing apoptosis in the mature CNS.
Spinal muscular atrophy (SMA) is a hereditary neurodegenerative disorder characterized by a severe depletion of motor neurons in the spinal cord and brain stem. Many of the motor neurons observed at autopsy in SMA spinal cords display such features as chromatolysis which are consistent with the apoptosis. During maturation of the spinal cord, as many as 50% of motor neurons undergo apoptosis. This has led to the suggestion that a genetic defect in a neuronal apoptotic pathway may be responsible for motor neuron depletion in SMA. Recently two candidate genes, i.e., survival motor neuron (smn) and neuronal apoptosis inhibitory protein (naip), have been identified. The product of the naip gene was termed neuronal apoptotic inhibitory protein (NAIP) because of sequence homology with two baculoviral proteins (Cp-IAP and Op-IAP) that block virally induced apoptosis.
Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by a loss of nigrostriatal neurons which results in a severe depletion of dopamine (DA) levels in the basal ganglia. Rats which have sustained unilateral lesions of the nigrostriatal pathway produced by the catecholamine-specific neurotoxin, 6-hydroxydopamine (6-OHDA) serve as an animal model of PD. Unilateral injection of 6-OHDA into the medial forebrain bundle or the substantia nigra pars compact results in a rapid degeneration of the nigrostriatal pathway. However, injection of 6-OHDA into the striatum produces a progressive degeneration (>1 week) of the nigrostriatal pathway which is believed to more closely resemble the natural pathology of PD (Sauer and Oertel, 1994). No good treatment for the prevention of PD degeneration currently exists.
Epilepsy is characterized by brain seizures and often results in neural cell death. It has been observed that previously kindled rats, i.e., those rendered “epileptic” via daily application of low intensity electrical stimulation, show considerably less brain damage from kainic-acid induced status epilepticus as compared to naive rats. Status epilepticus (SE) is characterized by continuous electrographic and/or behavioral seizures that occur unabated for greater than 30 minutes. In naive, nonkindled rats, this condition ultimately results in severe neuronal damage in several brain regions. Despite experiencing a form of SE as severe as that observed in a naive rat, the kindled “epileptic” rats show only minimal neuronal loss. Determining the mechanisms responsible for this experience-induced neuroprotection could provide novel approaches to the amelioration of brain damage resulting not only from SE, but also from other neurological traumas such as stroke.
Ischemia results when blood flow to the CNS is interrupted. This is frequently what happens following traumatic injury and stroke. Cell death often results. If this interruption of blood flow effects a large area of the CNS, or lasts for a long period of time, death due to loss of neurological function required for viability occurs. However, if blood flow to the CNS is transiently interrupted and recirculation is established within minutes, only certain neurons in the brain will die.
The best experimental model partial neuronal death due to ischemia is the 4-vessel occlusion model. In this global model of cerebral ischemia, neurons in neocortical layers 3, 5, and 6′, small- and medium-sized striatal neurons; and hippocampal CA1 pyramidal neurons are among the most vulnerable (Pulsinelli et al., Ann. Neurol. 11:491-498, 1982). By contrast, cholinergic interneurons in the striatum and CA3 pyramidal neurons in the hippocampus are more resistant to the damaging effects of transient global ischemia (Francis and Pulsinelli, Brain Res. 243:271-278, 1982).
SUMMARY OF THE INVENTION
We have discovered that increased levels of NAIP and IAP polypeptides used provide neuroprotection and allow neural regeneration.
We have found that increased NAIP levels correlate with neuronal survival under a variety of conditions which normally result in neuronal cell death. Furthermore, increased NAIP and IAP levels allow axonal regrowth after axotomy.
Taken together, these findings indicate that NAIP and the IAPs play a key role in conferring resistance to ischemic damage and neural degeneration, and allowing neural repair. Accordingly, our discovery provides both methods for the prevention of neural damage and neural repair and methods by which to screen for neuroprotective and neuroregenerative compounds.
The invention may be summarized as follows.
In the two principle aspects, the invention provides a method for inhibiting death of a cell of the nervous system and/or enhancing neural regeneration. The methods include increasing the biological activity (e.g., levels or neuroprotective effects as described herein) of a polypeptide selected from the group consisting of the NAIP or an TAP. This increasing in cells exposed or likely to be exposed to ischemic conditions is part of the invention if it is sufficient to produce a 20% or greater increase in the likelihood that a cell will survive following an event which normally causes at least a degree of nerve cell death. In some embodiments, the polypeptide is mammalian NAIP, HIAP, HIAP2, or XIAP. Most preferably the polypeptide is NAIP. In one preferred embodiment the polypeptide is a NAIP polypeptide lacking a portion of the carboxy-terminus. In a related embodiment the nerve cell is the CNS, and most preferably, the cell is a neuronal cell known to be susceptible to post-ischemic cell death.
In another embodiment the increasing is by administration of a transgene encoding the NAIP or TAP polypeptide in an expressible genetic construct. The transgene may be in a construct which includes various types of promoters, e.g., a constitutive promoter, a neurofilament promoter, or a regulatable promoter.
The transgene may be in a viral vector, e.g., an adenovirus vector, a herpes virus vector, or a polio virus vector. In preferred embodiments, the transgene enco
Crocker Stephen J.
Korneluk Robert G.
MacKenzie Alexander E.
Robertson George S.
Xu Daigen
Aegera Therapeutics Inc.
Bicker-Brady Kristina
Clark & Elbing LLP
Falk Anne-Marie
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