ADENO-ASSOCIATED VIRUS (AAV)-MEDIATED NGF GENE-TRANSFER INTO PC12 CELLS AND RAT BRAIN
S. Wang, W.J. Millard, E.M. Meyer
Dept. of Pharmacology, Dept. of Pharmacodynamics, University of Florida Colleges of Medicine and Pharmacy, Gainesville, FL 32610
An early pathological feature of Alzheimer's Diesease is the loss of basal
forebrain cholinergic neurons. Exogenous nerve growth factor (NGF) delivered
intracerebroventricularly can promote the survival of these neurons in vivo,
but administered peripherally does not cross the blood brain barrier. Another approach to increase NGF levels in the CNS involves gene transfer;
in this approach, a single injection may be sufficient to effect responses for indefinite
periods of time. AAV-derived plasmids have been used in this laboratory to
drive expression of neuropeptide Y mRNA and peptide in primary and neuronal cell lines,
and to induce feeding behavior when injected into the hypothalamus. To test
whether this system would permit expression of trophic factors, several
NGF-containing plasmids were constructed: 1) NGF regulated by a CMV promoter
with AAV-terminal repeats (pTRngf), 2) NGF regulated by the endogenous AAV p40 promoter with AAV terminal repeats (dlkngf), and 3) NGF regulated by the CMV
promoter, with no AAV-terminal repeats (pcngf). PC12 cells transfected with each construct ceased to divide and began to extend neurite-like processes
within 24 hours of transfection. Expression appeared to be transient with
apparent loss of neurites 7 days post-transfection. Unilateral injection of
pTRngf encapsulated by reconstituted Sendai viral envelopes into rat septum,
followed one week later by ipsilateral electrolytic fimbrial lesions resulted
in choline acetyltransferase levels that were 162% and 119% of unlesioned control value in
septum and hippocampus, though neither value reached statistical significance (n= 3-4).
Additional studies pertaining to relative promoter activity, duration of mRNA
and peptide expression, and cell survival will be described. (SW is supported
by training grant NS-07333)