A microRNA embedded AAV alpha-synuclein gene silencing vector for dopaminergic neurons

Abstract

Alpha-synuclein (SNCA), an abundantly expressed presynaptic protein, is implicated in Parkinson disease (PD). Since over-expression of human SNCA (hSNCA) leads to death of dopaminergic (DA) neurons in human, rodent and fly brain, hSNCA gene silencing may reduce levels of toxic forms of SNCA and ameliorate degeneration of DA neurons in PD. To begin to develop a gene therapy for PD based on hSNCA gene silencing, two AAV gene silencing vectors were designed, and tested for efficiency and specificity of silencing, as well as toxicity in vitro. The same hSNCA silencing sequence (shRNA) was used in both vectors, but in one vector, the shRNA was embedded in a microRNA backbone and driven by a pol II promoter, and in the other the shRNA was not embedded in a microRNA and was driven by a pol III promoter. Both vectors silenced hSNCA to the same extent in 293T cells transfected with hSNCA. In DA PC12 cells, neither vector decreased expression of rat SNCA, tyrosine hydroxylase (TH), dopamine transporter (DAT) or the vesicular monoamine transporter (VMAT). However, the mir30 embedded vector was significantly less toxic to both PC12 and SH-SY5Y cells. Our in vitro data suggest that this miRNA-embedded silencing vector may be ideal for chronic in vivo SNCA gene silencing in DA neurons.

shRNA targeting α-synuclein prevents neurodegeneration in a Parkinson’s disease model

“…An alternative interpretation is that the neurodegeneration reported previously may have been attributable to factors other than loss of α-synuclein. This is compatible with other work showing that an AAV-H1:shRNA vector targeting human, but not rat, SNCA was toxic in the rat substantia nigra (as was a similar vector expressing shRNA targeting luciferase), suggesting that nonspecific toxicity from shRNA expression at high levels may be important (67,68). Regardless, the absence of neurodegeneration resulting from α-synuclein knockdown in our study is compatible with findings in a range of other model systems (28,29,45,58,61,63).…”

Caspase-3 and GFAP as early markers for apoptosis and astrogliosis in shRNA-induced hippocampal cytotoxicity

“…We suggest that the design of shRNA-based studies should involve careful consideration of several parameters, such as shRNA dose (Grimm, 2011), promoter choice (Lebbink et al, 2011;Sun et al, 2013), AAV serotype (Ehlert et al, 2010) and construct backbone (Boudreau et al, 2009;Han et al, 2011;McBride et al, 2008). These factors should subsequently be tested with respect to titer, construct and specificity in vivo.…”

Gene Therapy for Misfolding Protein Diseases of the Central Nervous System

“…Unfortunately, AAV-shRNA expression did not protect against nigral neuronal loss in these animals and, when injected alone, AAV-shRNA expression alone resulted in dopaminergic cell loss in the SN [90]. Interestingly, embedding the shRNA used in the latter study into a microRNA resulted in reduced toxicity in cell cultures, but still achieved a significant silencing of hα-syn (up to 60 % less hα-syn expression) [91]. Unfortunately, and like the comments made earlier about AD models, rodents engineered to overexpress hα-syn are not good models of PD and, probably, any pathology corresponds more to the toxic effect of hα-syn overexpression per se.…”