HSV MEDIATED TRANSFER OF THE HPRT GENE TO THE CENTRAL NERVOUS SYSTEM

  • Evans, Christopher H (PI)
  • Fink, David J. (PI)
  • Goss, James (PI)
  • Goins, William F. (PI)
  • Glorioso, Joseph C. (PI)
  • Deluca, Neal A. (PI)
  • Robbins, Paul D. (PI)
  • Barranger, John (PI)
  • Lazo, John (PI)
  • Deluca, Neal A. (PI)
  • Watkins, Simon (PI)
  • Huang, Leaf (PI)
  • Robbins, Paul D. (PI)
  • Barranger, John (PI)
  • Lazo, John (PI)
  • Deluca, Neal A. (PI)
  • Huang, Leaf (PI)
  • Robbins, Paul D. (PI)
  • Fink, David J. (PI)
  • Yoshimura, Naoki (PI)
  • Glorioso, Joseph C. (PI)
  • Deluca, Neal A. (PI)
  • Huang, Leaf (PI)
  • Robbins, Paul D. (PI)
  • Watkins, Simon (PI)
  • Goins, William F. (PI)

Project: Research project

Project Details

Description

The goal of this research is to develop HSV as a versatile and efficient
gene transfer system. Gene transfer and expression of the HPRT gene will
be used as a model system toward the long-term goal of gene therapy for
Lesch-Nyhan disease. The project is organized into six interrelated
aims: (i) Attempts will be made to engineer a completely defective
nontoxic HSV vector backbone by deletion of genes which are expressed
immediately on infection. The virus mutants will be propagated on
complementing cell lines and characterized for the effects on neuronal
cells in culture following infection. Parameters to be considered are
virus multiplicity, and host cell macromolecular synthesis and viability.
(ii) Nontoxic vectors (NTV) will be studied for their ability to persist
long term in cell culture as extrachromosomal elements and for
integration events using selectable markers. The expression of viral
genes will be monitored including the latency associated transcripts
using Northern and RT-PCR approaches. (iii) We attempt to direct and
detect homologous segment of the integration events involving a mutant
HPRT gene cloned into HSV and the wild-type HPRT gene resident in B103
cells. (iv) The HSV-NTV will be stereotactically inoculated into
hippocampus and basal ganglia and examined for neuronal cell damage using
histochemical approaches. Both nonimmune and HSV-immune animals will be
examined to evaluate possible immunocytotoxic responses. Genome
persistence and expression of viral latent and lytic genes will be
examined by in situ hybridization and RT-PCR. (v) A series of experiments
will be carried out to exploit the latency active promoter (LAP), strong
non-HSV promoters or recombinant promoters for expression of the lacZ
long-term in vitro and in vivo. Moreover, attempts will be made to
design recombinant promoters responsive to the potent Gal4-VP16
transactivator which auto stimulates both its own expression and lacZ
expression in the same viral genome. (vi) The HSV-NTV with a suitable
promoter system developed in experiments above will be exploited to
express the HPRT gene in the brains of HPRT deficient mice. Gene
expression will be characterized by in situ hybridization, and enzyme
production and activity in brain extracts by immuno- and biochemical
assays.
StatusFinished
Effective start/end date6/1/937/31/12

Funding

  • National Institute of Diabetes and Digestive and Kidney Diseases
  • National Institute of Diabetes and Digestive and Kidney Diseases
  • National Institute of Diabetes and Digestive and Kidney Diseases
  • National Institute of Diabetes and Digestive and Kidney Diseases
  • National Institute of Diabetes and Digestive and Kidney Diseases
  • National Institute of Diabetes and Digestive and Kidney Diseases

ASJC

  • Medicine(all)

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