Transduction of Brain Dopamine Neurons by Adenoviral Vectors Is Modulated by CAR Expression: Rationale for Tropism Modified Vectors in PD Gene Therapy

Gene-based therapy is a new paradigm for the treatment of Parkinson disease (PD) and offers considerable promise for precise targeting and flexibility to impact multiple pathobiological processes for which small molecule agents are not available. Some success has been achieved utilizing adeno-associated virus for this approach, but it is likely that the characteristics of this vector system will ultimately create barriers to progress in clinical therapy. Adenovirus (Ad) vector overcomes limitations in payload size and targeting. The cellular tropism of Ad serotype 5 (Ad5)-based vectors is regulated by the Ad attachment protein binding to its primary cellular receptor, the coxsackie and adenovirus receptor (CAR). Many clinically relevant tissues are refractory to Ad5 infection due to negligible CAR levels but can be targeted by tropism-modified, CAR-independent forms of Ad. Our objective was to evaluate the role of CAR protein in transduction of dopamine (DA) neurons in vivo.Ad5 was delivered to the substantia nigra (SN) in wild type (wt) and CAR transgenic animals. Cellular tropism was assessed by immunohistochemistry (IHC) in the SN and striatal terminals. CAR expression was assessed by western blot and IHC. We found in wt animals, Ad5 results in robust transgene expression in astrocytes and other non-neuronal cells but poor infection of DA neurons. In contrast, in transgenic animals, Ad5 infects SNc neurons resulting in expression of transduced protein in their striatal terminals. Western blot showed low CAR expression in the ventral midbrain of wt animals compared to transgenic animals. Interestingly, hCAR protein localizes with markers of post-synaptic structures, suggesting synapses are the point of entry into dopaminergic neurons in transgenic animals.These findings demonstrate that CAR deficiency limits infection of wild type DA neurons by Ad5 and provide a rationale for the development of tropism-modified, CAR-independent Ad-vectors for use in gene therapy of human PD

Functional expression of Rab escort protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo

Choroideremia (CHM) is an X-linked retinal degeneration of photoreceptors, the retinal pigment epithelium (RPE) and choroid caused by loss of function mutations in the CHM/REP1 gene that encodes Rab escort protein 1. As a slowly progressing monogenic retinal degeneration with a clearly identifiable phenotype and a reliable diagnosis, CHM is an ideal candidate for gene therapy. We developed a serotype 2 adeno-associated viral vector AAV2/2-CBA-REP1, which expresses REP1 under control of CMV-enhanced chicken β-actin promoter (CBA) augmented by a Woodchuck hepatitis virus post-transcriptional regulatory element. We show that the AAV2/2-CBA-REP1 vector provides strong and functional transgene expression in the D17 dog osteosarcoma cell line, CHM patient fibroblasts and CHM mouse RPE cells in vitro and in vivo. The ability to transduce human photoreceptors highly effectively with this expression cassette was confirmed in AAV2/2-CBA-GFP transduced human retinal explants ex vivo. Electroretinogram (ERG) analysis of AAV2/2-CBA-REP1 and AAV2/2-CBA-GFP-injected wild-type mouse eyes did not show toxic effects resulting from REP1 overexpression. Subretinal injections of AAV2/2-CBA-REP1 into CHM mouse retinas led to a significant increase in a- and b-wave of ERG responses in comparison to sham-injected eyes confirming that AAV2/2-CBA-REP1 is a promising vector suitable for choroideremia gene therapy in human clinical trials. © 2013 The Author(s)

Specific patterns of defective HSV-1 gene transfer in the adult central nervous system: implications for gene targeting.

Viral vectors are a means by which genes can be delivered to specific sites in the adult central nervous system. Nevertheless, the interaction between the viral vector and cells of the nervous system, which forms the basis for specific gene transfer, is not well understood. In this study a nonreplicating defective herpes simplex virus type 1 vector, expressing the marker gene lacZ, was stereotaxically injected at varying titers into the rat central nervous system. Three sites were targeted: the caudate nucleus, dentate gyrus, and cerebellar cortex, and the resulting patterns of beta-galactosidase activity were examined. Many cells of neuronal and glial morphology, and of differing neuronal subtypes, expressed beta-galactosidase at each of the injection sites. However, beta-galactosidase activity was also detected in distant secondary brain areas, the neurons of which make afferent connections with the primary sites. This strongly suggested that the retrograde transport of defective virus was the basis for the enzyme activity observed at a distance. Moreover, retrograde transport to secondary sites was found to be highly selective and restricted to certain retrograde neuroanatomical pathways in a specific and titer dependent fashion. The pathways observed were predominantly, but not exclusively, monoaminergic in origin. This finding is supported by reports of specific tropism by HSV for monoaminergic circuits in experimental encephalitis and transneuronal tracing studies. Our observations suggest that certain functional neuronal populations, which are permissive for the retrograde transfer of defective HSV-1 vectors, might be specifically targeted for gene transfer using this approach. Conversely, a knowledge of the pathways permissive for viral uptake, retrograde transfer, and subsequent gene expression will be essential in order to predict the consequences of gene transfer using viral vectors