Here we report a comprehensive characterization of a new model of HD generated by AAV1/2 vector-mediated transfer of N-terminal htt constructs to the rat striatum. Recombinant AAV has emerged as a vector of choice for gene transfer to the CNS, due to its strong neuronal tropism and lack of pathogenicity in mammals [5
]. The packaging capacity of 4.7kb precludes AAV vector-mediated over-expression of full-length htt, however N-terminal truncated htt constructs have been successfully used previously to recapitulate elements of HD in experimental animals [8
]. Moreover, there is growing evidence to support a ‘toxic fragment hypothesis’, whereby neuropathological events in HD are triggered by proteolytic cleavage of an N-terminal htt fragment [2
]. The AAV-HD70 model recapitulates some critical elements of HD and bears both similarities and differences to several previously reported genetic models of HD. The rapidly progressive phenotype most closely resembles previous models generated by virus vector-mediated gene transfer [7
]. The extent of striatal neuronal cell loss observed was greater than previously reported in earlier viral vector-based models of HD in rats [7
] and similar to that recently reported using AAV1/8 in mice by DiFiglia et al. [10
] as a consequence of the increased expression achieved using AAV1/2 vectors, both in terms of expression level and the volume of striatal transduction. Moreover, the striatal neuronal cell loss observed as early as 5 weeks post-injection of AAV-HD70 was over twice that reported for 12 month old YAC128 mice that exhibit the greatest extent of striatal neuronal cell loss of all the transgenic HD mouse models [29
]. Differences in the rapidity of disease progression correlate to some degree with mutant htt expression level in genetic models of HD. Indeed, analysis of the temporal expression profile by RT-PCR revealed a dramatic level of N-terminal htt expression resulting from AAV vector-mediated gene transfer. This may prove to be advantageous in that it may facilitate more rapid in vivo
screening of therapeutics than is currently available using transgenic models.
Interestingly, we found that AAV1/2 vector-mediated transduction patterns influenced the neuropathological phenotype in our model. First, it was found that AAV1/2 transduction of striatal neurons is non-uniform, most notably with increased transduction of cholinergic interneurons compared to other neuronal populations. The reason for this is unclear, however it may be that cholinergic interneurons express different cell-surface receptors that facilitate enhanced tropism through more rapid internalization of AAV1/2 virions. Entry of AAV into cells is dependent upon binding to cell surface glycosaminoglycan receptors and other co-receptors, although the mechanisms underlying selective tropisms of AAV serotypes remain largely unknown [30
]. Moreover, differences in intra-cellular processing of AAV vectors may play a role in transduction of various neuronal populations [30
]. Over 100 serotypes of AAV have been isolated to date, and characterization of novel AAV serotypes revealed different patterns of transduction in a diverse array of tissues [31
]. Moreover, it has been shown that transduction patterns in the nigro-striatal system can vary greatly between different AAV serotypes [19
]. Further investigation to elucidate differences in AAV tropism and transduction among neuronal populations in the striatum and other brain regions may lead to improved modeling of neurodegenerative diseases in experimental animals. Secondly, we found that neuronal transduction by AAV1/2 vectors was altered by changing the transgene alone. It has been proposed that changes in secondary structure of AAV vector genomes containing different constructs may alter tropism by changing the conformation of capsid proteins on the virion surface, leading to altered interactions with cell surface receptors [35
]. This hypothesis awaits verification by structural studies of recombinant AAV vectors.
Axonal vector transport also played a role in the neuropathological phenotype in our model. Robust expression of AAV-HD70 was observed in the GP and SN following striatal injection of AAV vectors, leading to toxicity in distal structures of the basal ganglia. These results confirm previous reports that retrograde transport can lead to transduction of neurons in brain regions distal to the site of injection [20
], but also indicate that neuronal cell loss in distal brain regions can result from expression of neuro-toxic gene constructs such as HD70. Although the primary neuropathology in HD occurs in the striatum, neurodegeneration and atrophy of the GP and SNpr is also observed in HD brains, however neuronal cell loss in the SNpc is less typical of HD [14
]. Taken together, these results raise some issues regarding the use of viral vectors for modeling neurodegenerative diseases that warrant caution and require to be addressed. For example, these findings indicate that the use of viral vector systems such as AAV could potentially introduce artifacts into functional genomics and disease modeling studies.
Nevertheless, the robust striatal neurodegenerative phenotype observed in the AAV-HD70 model suggests that it is well-suited for the rapid screening of neuroprotective strategies for HD. Here, we demonstrated for the first time that AAV vector-mediated RNAi was highly effective as a neuroprotective therapy in an animal model of HD. Our findings are in agreement with previous reports supporting the utility of RNAi as a therapeutic approach for HD and other polyQ expansion diseases in cell culture [36
] and transgenic mouse models [11
]. Recently, DiFiglia et al. also reported that a cholesterol-conjugated siRNA targeting human htt mRNA inhibited expression of the AAV1/8Htt
100Q vector and led to increased survival of striatal neurons and improved motor behaviors in mice [10
]. Importantly, however, siRNA-mediated silencing is transient and would require frequent repeat interventions for treating HD patients. In contrast, AAV vector-mediated delivery of shRNAs offers a long-term approach for therapeutic disease gene silencing following a single injection [40
Despite the clear therapeutic potential of RNAi-based gene therapy for HD, several issues are challenging translation towards the clinic, including allele-specific silencing, ‘off-target effects’ and potential toxicity [41
]. Despite these caveats, RNAi may prove to be an effective therapeutic strategy for HD and other neurodegenerative diseases. Our results support AAV vector-mediated knockdown of mutant htt expression as a direct approach to preventing striatal neurodegeneration and associated behavioral impairment. Translation of viral vector-mediated RNAi to a non-human primate genetic model of HD [9
] would provide further opportunity for pre-clinical assessment of the safety and efficacy of this therapeutic approach.