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).

Delayed delivery of AAV-GDNF prevents nigral neurodegeneration and promotes functional recovery in a rat model of Parkinson's disease

“…In contrast, rats injected with AAV-LacZ in the striatum demonstrated no ␤-galactosidase signal in the SN. In agreement with the previous study, 32 AAV vector per se was not retrogradely transported in the central nervous system, but GDNFflag protein was retrogradely transported from the striatum 387 to the DA neurons in the SN. We also detected substantial expression of GDNFflag protein in the striatum at 20 weeks after injection.…”

Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes

“…We determined, based on cell morphology and double-labeling to NeuN (Figure 3d, e; Figure 4e), that Ͼ99% of cells transduced with the NSE promoter cassette were neurons. Using the 1.8 kb NSE promoter, we also observed striking anterograde transport, previously reported by others, 10 when the vector was injected into the hippocampus; the GFP protein was expressed at high levels in well-defined neuronal layers of the contralateral hippocampus ( Figure 4f). With the EF promoter, we also found that the vast majority of cells transduced were neuronal (Figure 3b, c; Figure 4b, c) as defined by NeuN or MAP-2 expression.…”

“…In contrast, the rat NSE 1.8 kb promoter gave the highest level of luciferase activity in vitro and in vivo, suggesting that (along with chicken ␤-actin) it is the best promoter among those we tested to drive neuronal expression in the brain with AAV-2. One recent study using an AAV-2 construct with CMV promoter and SV40 polyA to drive a GFP reporter gene 10 found that ෂ50 nl of vector transduced 'hundreds of neurons' at the injection site. At best this corresponds to Ͻ10 000 neurons/l, which is over an order of magnitude less than observed with this optimized vector system.…”

“…The main approach for optimizing vectors such as adeno-associated virus (AAV-2) has traditionally been at the nucleic acid level, involving promoter and other gene regulatory elements, although recent work on viral subtypes or pseudotypes suggests that capsid modifications may be equally important in increasing expression or modifying the tropism of recombinant virus. [1][2][3][4] To date, most studies on brain gene transfer have focused on vectors carrying viral promoters, [5][6][7][8][9][10] although some have looked at mammalian cell type-specific promoters. [11][12][13][14][15][16][17] In this study, we compared the strength and stability of expression, as well as cellular specificity, of AAV-2 vectors expressing reporter genes under the control of constitutive, viral, or other cellular promoters.…”

Body weight is regulated by the brain: a link between feeding and emotion

“…This attempt will include the use of other transgenic mice expressing Cre in selected neuronal populations, as well as of stereotaxic injections of an adenoassociated viral vector that drive the Cre expression. 142 Clearly, these combined approaches will continue to evolve strategies to survey the physiologically important CNS circuitries mediating the effects of key metabolic signals including leptin, glucose, and ghrelin.…”