Differential adenoassociated virus vector-driven expression of a neuropeptide Y gene in primary rat brain astroglial cultures after transfection with Sendai virosomes versus Lipofectin.

The ability of Sendai virosomes or Lipofectin to introduce an AAV vector into primary rat brain astroglial cultures was characterized. The pJDT95npy vector was constructed by inserting rat NPY cDNA downstream from the indigenous AAV p5, p19 and p40 promoters in pJDT95. Lipofectin-mediated transfection with pJDT95npy (10 micrograms) resulted in pronounced expression of several NPY mRNA species: p5-driven (3.3 kb), p19-driven (2.7 kb) and p40-driven (0.6, 0.8, 1.1, and 1.8 kb). Exposure to virosomally encapsulated pJDT95npy (50 or 100 ng) resulted in transient expression of some p40-driven mRNA species (0.8 and 1.8 kb). Neither method produced astroglia cells which synthesized mature NPY immunoreactivity. This demonstrates that an AAV-derived vector can drive gene expression in astroglia, that Sendai virosomes can infuse vectors into astroglia, but that the amount of DNA infused in this manner may limit long term expression.

Fusogenic properties of Sendai virosome envelopes in rat brain preparations.

Sendai virosomes were characterized with respect to their ability to bind to, fuse with, and introduce substances into several rat brain preparations. Encapsulation efficiency for Sendai virosomes was enhanced but binding to cerebral cortical P2 preparations was attenuated by addition of bovine brain phosphatidylcholine during reconstitution. A higher percentage of Sendai virosomes than phosphatidylcholine liposomes appeared to bind to, fuse with and subsequently deliver [14C]sucrose into osmotically labile pools of the P2 preparation. Fusogenic activity was estimated by measuring dequenching of fluorescently labelled N-NBD-phosphatidylethanolamine. More virosomally encapsulated [14C]sucrose was bound to the P2 fraction than introduced into osmotically labile organelles, and the fraction of vesicles undergoing fusion was intermediate between these two values. Non-encapsulated [14C]sucrose did not bind to and was not taken up by the P2 fraction in a quantifiable manner. Virosomal envelopes also bound to primary cultures of rat brain neurons and glia in an apparently saturable manner. Addition of increasing amounts of the adenoassociated virus-derived vector pJDT95 increased encapsulation efficiency, and virosomes reconstituted in the presence of 60 micrograms DNA retained most of their binding activity (5.4% of total label) compared to those containing [14C]sucrose alone (8.4%). These data indicate that Sendai virosomes may be useful in the delivery of substances into brain-derived tissues, potentially for the modulation of gene expression and neurotransmission.