1992 |
Mccown, Thomas J. |
R55Activity Code Description: Undocumented code - click on the grant title for more information. |
Role of Inferior Collicular Cortex in Seizures @ University of North Carolina Chapel Hill
This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.
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0.958 |
1997 — 2011 |
Mccown, Thomas J. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Gene Therapy and Seizures @ University of North Carolina Chapel Hill
[unreadable] DESCRIPTION (provided by applicant): Theoretically, viral vector gene therapy holds great promise for the treatment of epilepsy, where in vivo expression of foreign genes could potentially suppress focal seizure sensitivity. Neurotransmitter receptors and ion channels offer obvious gene therapy targets, but a successful outcome is dependent upon the pattern of viral Vector transduction which to date cannot be predicted a priori. One means to circumvent this potential impediment would be to express an endogenous inhibitory neuropeptide that is subsequently secreted from the transduced cell. Two endogenous peptides, galanin (GAL) and neuropeptide Y (NPY), both can potently suppress limbic seizure activity. Recent preliminary findings with adeno-associated virus vectors (AAV) show that the secretion signal sequence for the laminar protein, fibronectin, can secrete vector-derived gene product in vitro and when placed in front of the GAL coding sequence, suppresses in vivo focal seizure sensitivity in a regulatable fashion and attenuates kainic acid-induced cell death in the hippocampus. Therefore, the present proposal will test the hypothesis that in vivo secretion of vector derived GAL or NPY will suppress acute seizure activity, reduce kainic acid seizure associated cell death and retard the spontaneous seizure activity that develops after pilocarpine administration. First, recombinant AAV vectors containing fibronectin secretory sequence in front of GAL or NPY coding sequences will be infused into the inferior colliculus, and both the persistence of seizure suppression and the ability to regulate gene expression will be evaluated. Then, these same recombinant AAV vectors will be tested in the kainic acid seizure model, evaluating if transduction of hippocampal hilar neurons can alter acute seizure sensitivity and seizure-induced cell damage. Subsequently, it will be determined if these AAV vectors can prevent the development of spontaneous seizures following pilocarpine-induced seizures. The findings from these studies could lead to a novel gene therapeutic approach to the treatment of epilepsy.
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0.958 |
2007 — 2008 |
Mccown, Thomas J. |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Directed Evolution of Adeno-Associated Virus Vectors For Seizure Gene Therapy @ University of North Carolina Chapel Hill
[unreadable] DESCRIPTION (provided by applicant): Epilepsy afflicts approximately 2.5 million people in the U.S., making epilepsy one of the most prevalent neurological disorders (Hauser and Hesdorffer, 1990). Although current, anti-epileptic medication effectively controls the seizures in approximately 70% of this population, for the remaining 30% medications do not adequately control the seizures (Kwan and Brodie, 2000). In these intractable cases, surgical resection offers a final option, and for those patients that qualify for surgery, up to 66% remain seizure free for 2 years post- surgery (Spencer et al., 2005). However, longer term prognosis is more uncertain, and if the site of seizure genesis impinges upon brain sites associated with critical functions, resective surgery is not a viable option (McIntosh et al., 2004; Ojemann, 1997). Recently, several studies have established gene therapy approaches that significantly influence seizure sensitivity in vivo. Using adeno-associated virus (AAV) vectors, the expression and constitutive secretion of the neuroactive peptide, galanin, can prevent seizure activity in vivo (Haberman et al., 2003; McCown, 2006), while the expression of prepro-neuropeptide Y also significantly attenuates seizure activity in vivo (Richichi et al., 2004). At present, though, the clinical application of these viral vector gene therapies will require direct neurosurgical injection into the brain. Recent developments in AAV directed evolution provide a potential means to circumvent neurosurgical intervention. Maheshri et al. (2006) showed that by introducing random changes in the AAV 2 capsid by error prone PCR, subsequent directed evolution resulted in AAV mutants that lacked heparin binding or evaded the in vivo immune response to AAV 2. In preliminary studies, the Samulski laboratory has shown that the combination of DNA shuffling techniques with directed evolution produces novel mutant AAV vectors with unique in vivo tropisms. Because seizures compromise the blood-brain barrier in areas of seizure activity, we propose to develop a mutant AAV library through DNA shuffling of AAV capsid 1-9 sequences. Then after kainic acid-induced seizures, the mutant library will be administered i.v. and through directed evolution techniques, novel mutant AAV vectors will be identified that following seizure activity, cross the compromised BBB and transduce neurons. The successful creation of such mutant AAV vectors could revolutionize the treatment of intractable seizure disorders. The proposed studies focus upon developing a novel gene therapy method for the treatment of neurological disorders. Using directed evolution techniques, we will select specific gene delivery vehicles that can access a seizure-impaired brain after peripheral intravenous administration. If successful, this novel approach could greatly advance the application of gene therapy to the treatment of neurological disorders. [unreadable] [unreadable] [unreadable] [unreadable]
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0.958 |
2013 — 2017 |
Mccown, Thomas J. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Development of Intravenous Aav Vectors For Intractable Epilepsy @ Univ of North Carolina Chapel Hill
DESCRIPTION (provided by applicant): Epilepsy afflicts approximately 3 million people in the U.S., and although current, anti-epileptic medication effectively controls the seizures in approximately 70% of this population, medications do not adequately control seizures for the remaining 30% (Kwan and Brodie, 2000). Because fewer than 10% of patients with drug refractory epilepsy are considered for surgical resection, a substantial number of epilepsy patients essentially have no effective therapeutic options (Engel et al.; 1992, Siegel, 2004). Even with the introduction of many new drugs, the number of drug resistant epilepsies has not decreased, prompting Loscher and Schmidt (2011) to state that the available evidence indicates that the efficacy and tolerability of drug treatment of epilepsy has not substantially improved. Recent studies by Gray et al (2010) have provided a potential solution to this problem. Using an acute limbic seizure model Gray et al. (2010) showed that capsid DNA shuffling and directed evolution could identify a novel, chimeric adeno-associated virus (AAV) clone (#83) which upon intravenous administration selectively crossed the acute seizure compromised blood-brain barrier and transduced cells in the CNS. In order to realize the full potential of this approach we hypothesize that additional DNA shuffling and directed evolution in a chronic limbic seizure model will produce safe, therapeutically effective, chimeric vectors. Two new brain specific AAV capsid libraries will be constructed, one based upon error prone PCR of clone 83 and the other composed of unique, multiple clones that cross the seizure compromised blood-brain barrier. The libraries will be injected intravenously into rats with documented chronic, spontaneous seizure activity. Subsequently, neurons will be dissociated from the hippocampus and the piriform cortex, and the mutant clones will be rescued. After in vivo validation of both transduction efficacy and peripheral biodistribution, the most effective clones will be packaged with proven therapeutic neuropeptide cassettes and recombinant virus will be produced. These novel vectors will be administered intravenously to rats with documented spontaneous seizure activity in order to assess the ability to attenuate spontaneous limbic seizure activity, as well as after acute seizures to test for anti-epileptogenic actions. If successful the findings would dramatically shift current epilepsy treatment paradigms and significantly impact the treatment refractory epileptic population.
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0.946 |
2021 |
Mccown, Thomas J. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Aav Capsid-Promoter Interactions Determines Cns Cell Selective Gene Expression in Vivo @ Univ of North Carolina Chapel Hill
ABSTRACT Adeno-associated virus (AAV) vectors occupy a prominent role in recent CNS clinical trials particularly with respect to the use of AAV serotype 9 (AAV9) for single gene genetic disorders, such as spinal muscular atrophy and giant axon neuropathy (Mendell et al., 2018; Bailey et al., 2018). Given the need for selective cellular transduction, capsid modification, cell specific promoters and cell specific enhancers all have demonstrated success in achieving specific vector properties (Asokan et al., 2012; Dimidschstein et al., 2016; Grimm and Buning, 2017). In a recently accepted manuscript, we established a previously unknown interaction between the AAV9 capsid and different constitutive promoters, namely the ability to directly influence cell specific gene expression in the CNS. Using identical transgenes and the AAV9 capsid, CBA promoter driven gene expression exhibited a dominant neuronal gene expression in the rat striatum, but when gene expression was driven by the truncated Cbh promoter, gene expression was significantly shifted to striatal oligodendrocytes. Moreover, an AAV9 chimera containing six glutamate insertions after amino acid 139 in VP1/2 exhibited oligodendrocyte gene expression for both CBA and Cbh driven gene expression while a six alanine insertion in the same site reversed the Cbh driven gene expression back to neurons. Recently, preliminary findings revealed a similar AAV9 capsid interaction with the JetI synthetic promoter that influenced cellular gene expression in vivo. Given the highly novel nature of this capsid-promoter interaction, in vitro studies will define the mechanisms that underlie the glutamate and alanine shifts in cellular gene expression, including capsid conformation, intracellular trafficking, RNA splicing and VP1,2,3 interactions. In vivo studies will assess those promoter elements that contribute to the interactions, and specific AAV9 capsid elements that influence changes in in vivo cellular gene expression. Given the numerous applications of AAV9 vectors, the findings should significantly advance our understanding of basic capsid-promoter interactions and prove crucial to future design of AAV9 based gene therapies.
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0.946 |