ANTIEPILEPTIC EFFECT OF GENETICALLY MODIFIED FIBROBLASTS

Project: Research project

Description

The applicant's long term career-goal is to become a physician scientist
dedicated to neuroscience. The ultimate direction is to develop improved
methods of treating human epilepsy. The applicant plans to use a
combination of gene transfer and neural grafting as a tool to study
seizure propagation and as a potential therapeutic approach to epilepsy. Local application of GABA receptor agonist (e.g. muscimol) or GABA
transaminase inhibitor (e.g. gamma-vinyl GABA) into discrete brain regions
(e.g. substantia nigra, entopeduncular nucleus, amygdala and inferior
colliculus) could suppress seizure in various animal models of epilepsy.
Moreover, chronic infusion of GABA locally into motor cortex completely
blocked the behavioral and electrographical seizure in naturally
photosensitive baboon. The impermeability to blood-brain-barrier and
global toxicity, however, preclude the systemic use of these agents as
antiepileptic drugs. Therefore, the overall objective of this proposal is
to test the idea that grafting the genetically modified, GABA-secreting
fibroblasts into discrete brain regions can modify seizure manifestations.
To address this objective, glutamic acid decarboxylase (GAD) gene will be
used as the reporter gene, retroviruses will be the gene delivery vectors
and both primary skin fibroblasts and fibroblasts cell lines will be the
donor cells. The reasons for the use of fibroblasts as donor cells are
(1) retroviruses require dividing cells for infection; (2) fibroblasts are
readily available and easily cultured and manipulated in vitro; (3)
essential to the long-term strategy of potential therapy is the use of
primary skin fibroblasts as donor cells for autograft. Specifically, the proviral gene of wild-type retrovirus will be isolated
to construct the plasmid by replacing the genes necessary for viral
replication with the GAD gene. The transmissible but replication-
incompetent retroviral vectors carrying GAD gene can be produced by
introducing the constructed plasmid to the cells lines containing the
proviral gene of retroviral helper virus. By transfection with the
vectors, GAD gene will be integrated into fibroblasts. The in vitro
expression of GAD gene will be quantitated by measuring mRNA and secreted
GABA. To monitor the function in vivo, the genetically modified
fibroblasts will be grafted into hippocampus or lateral ventricle and
tested for the effects on the seizure phenomenon (behavioral and
electroencphalographical seizure), seizure threshold and seizure-induced
neuronal degeneration. The model systems for these purposes are a chronic
model of epilepsy induced by fimbria-fornix lesions and an acute model of
limbic seizure induced by intraamygdala injection of kainic acid. The
long-term graft survival, in vitro gene expression and host immune
reaction will be assessed by immunohistochemical approaches using
antibodies to various specific cell markers.
StatusFinished
Effective start/end date12/1/8911/30/95

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $91,584.00
  • National Institutes of Health
  • National Institutes of Health

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Anticonvulsants
Fibroblasts
Seizures
Glutamate Decarboxylase
Genes
Epilepsy
Retroviridae
gamma-Aminobutyric Acid
Plasmids
Entopeduncular Nucleus
Brain Fornix
Tissue Donors
Helper Viruses
Vigabatrin
GABA Agonists
Cell Line
Muscimol
Skin
Papio
Lateral Ventricles

ASJC

  • Medicine(all)
  • Neuroscience(all)