Recombinant adeno-associated virus vector: use for transgene expression and anterograde tract tracing in the CNS

Abstract

We used a recombinant adeno-associated virus vector (AAV) to deliver a foreign gene, green fluorescent protein (GFP), into mature neurons in adult rat CNS in vivo. Microinjections of AAV as small as 50 nl transduced hundreds of neurons at the injection site. There was virtually no retrograde transport as fewer than one neuron per brain was found distant from the injection site that exhibited GFP immunoreactivity. The gene product, GFP, filled the entire neuronal cytoplasmic compartment; GFP immunoreactivity was robust in cell bodies, axons, and nerve terminals. There was no tissue damage at the injection sites or pathogenicity indicated by changes in astrocytic or microglial markers. There was no inflammatory response as judged by leukocytic invasion. Gene expression in transduced cells was robust and apparently permanent: there was no evidence of phenotypic reversion up to 12 weeks following infection. AAV is an excellent vector for introducing foreign genes into mature CNS neurons. Not only might it be an ideal vehicle for gene therapy, but also the GFP-containing AAV presents a new strategy for tracing long axonal pathways in the CNS, which is difficult with current tracers (PHAL, biotinylated dextrans).

Fig. 1

Schematic of the Acp-UF5 vector. The following abbreviations are used: ITR—inverted terminal repeats, CMV—cytomegalovirus promoter, Xba1 and Sal1—restriction sites, SV40 pA—the simian virus 40 polyadenylation sequence.

Fig. 2

Digital photomicrographs of AAV injection site. Shown are ipsi- (A) and contralateral (A′) fields of an injection site stained with anti-GFP antiserum in case AAV10. An adjacent series of sections was stained for GFAP. B and B′ show the section adjacent to that shown in A and A′. Note that the GFAP staining in the external lateral subnucleus is always slightly greater than the other PB subnuclei. Staining was no different on the ipsi- (B) compared to contralateral (B′) to the AAV injection. Panel C shows microglial staining in another section through the injection site of the same brain. D shows staining for leukocyte common antigen (CD45). The insets in B, C, and D are higher magnification images of stained cells. The scale bar shown in the inset in D denotes 10 μm. A higher magnification of the boxed area in panel A is shown in Fig. 3.

Fig. 3

High magnification photomicrograph of AAV-GFP transduced neurons. The area shown is a higher magnification of the boxed area at the ventrolateral tip of the superior cerebellar peduncle in Fig. 2A. Note the Golgi-like filling of GFP immunoreactive cells of neuronal morphology (compare with insets in Fig. 2).

Fig. 4

High magnification photomicrographs of terminal fields of GFP-immunoreactive neurons. Terminal fields were located in the rostral ventrolateral medulla (A), spinal trigeminal nucleus (B), lateral hypothalamus (C), and spinal cord (D). Scale bar in A applies to A–C. Scale bar in D denotes 50 microns.

Fig. 5

Photomicrograph depicting GFP-IR terminal fields in the central nucleus of the amygdala. Note that nerve terminals form dense clusters surrounding target neurons (arrowheads and inset). The scale bar in the inset denotes 20 μm.