Novel AAV-mediated Therapeutic Strategies for Epilepsy Public Deposited

Downloadable Content

Download PDF
Last Modified
  • March 20, 2019
Creator
  • Foti, Stacey
    • Affiliation: School of Medicine, UNC Neuroscience Center, Neuroscience Curriculum
Abstract
  • Epilepsy afflicts 2.1 million people in the United States, and many patients develop drug resistant seizures. For these patients, gene therapy may be an attractive treatment option. Recent studies have shown that viral vector-mediated expression of neuropeptides such as galanin and neuropeptide Y (NPY) can attenuate seizure sensitivity and prevent seizure-induced cell death in the brain. NPY13-36 is a C-terminal peptide fragment of NPY that activates the NPY-Y2 receptor, thought to mediate the anti-seizure activity. Therefore we investigated if recombinant adeno-associated virus (AAV)-mediated expression and constitutive secretion of NPY or NPY13-36 could alter limbic seizure sensitivity. We found that AAV-mediated delivery of both NPY and NPY13-36 attenuates limbic seizures, and provides a vector technology platform for delivering therapeutic peptide fragments with increased receptor selectivity. To further explore the potential of this platform, we utilized a strategic approach to deliver multiple neuropeptides simultaneously. To achieve this, we used a proteolytic strategy such that our vectors contained a single promoter driving expression of a cleavable chimeric fusion protein of galanin and NPY13-36. The chimeric fusion protein contained a linker sequence capable of being cleaved by the intracellular protease furin, and also contained a fibronectin constitutive secretion signal (FIB) sequence. We first characterized several constructs in vitro, and determined that 1) a single FIB sequence was sufficient to cause secretion of both proteins, 2) the proteins were cleaved from one another regardless of position relative to the cleavage sequence, 3) cleavage efficiency of secreted proteins was 100%, 4) if the cleavage sequence was absent, uncleaved fusion protein was secreted into the medium. We then tested these constructs in a limbic seizure model, and showed that all of our vectors were capable of attenuating limbic seizure sensitivity. However, no additional efficacy was observed with the vector delivering both galanin and NPY13-36. Surprisingly, the level of attenuation was less than with previously published single peptide vectors, suggesting reduced translation efficiency. This body of work describes a gene therapy vector technology platform to express and constitutively secrete single and multiple proteins from transduced cells, and to deliver peptide fragments that are capable of selective receptor targeting.
Date of publication
Keyword
DOI
Resource type
Rights statement
  • In Copyright
Advisor
  • McCown, Thomas
  • Deshmukh, Mohanish
  • Lund, Pauline Kay
  • Samulski, Richard
  • Kafri, Tal
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2008
Language
Access
  • This item is restricted from public view for 1 year after publication.
Parents:

This work has no parents.

Items