An alpha-synuclein AAV gene silencing vector ameliorates a behavioral deficit in a rat model of Parkinson's disease, but displays toxicity in dopamine neurons

Abstract

Effects of silencing ectopically expressed hSNCA in rat substantia nigra (SN) were examined as a novel therapeutic approach to Parkinson’s disease (PD). AAV-hSNCA with or without an AAV harboring a short-hairpin (sh)RNA targeting hSNCA or luciferase was injected into one SN. At 9wks, hSNCA-expressing rats had reduced SN dopamine (DA) neurons and exhibited a forelimb deficit. AAV-shRNA-SNCA silenced hSNCA and protected against the forelimb deficit. However, AAV-shRNA-SNCA also led to DA neuron loss suggesting undesirable effects of chronic shRNA expression. Effects on nigrostriatal-projecting neurons were examined using a retrograde tract tracer. Loss of striatal-projecting DA neurons was evident in the vector injection site, whereas DA neurons outside this site were lost in hSNCA-expressing rats, but not in hSNCA-silenced rats. These observations suggest that high levels of shRNA-SNCA were toxic to DA neurons, while neighboring neurons exposed to lower levels were protected by hSNCA gene silencing. Also, data collected on DA levels suggest that neurons other than or in addition to nigrostriatal DA neurons contributed to protection of forelimb use. Our observations suggest that while hSNCA gene silencing in DA neurons holds promise as a novel PD therapy, further development of silencing technology is required.

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).…”

Recent Advances in RNA Interference Therapeutics for CNS Diseases

“…While shRNAs may have more potent silencing capability, they are often expressed at very high levels and can saturate the RNAi machinery, which disrupts endogenous miRNA processing and can induce toxicity [18,19]. Artificial miRNAs, however, are generally safer and less toxic, and they do not appear to disrupt endogenous miRNA processing [18][19][20][21][22][23]. Although artificial miRNAs are less toxic, their safety profile is also dictated by the design of the RNAi sequence.…”

“…[22,132] LRRK2 AS Allele-specific silencing of mutant α-syn and LRRK2 was achieved in vitro using artificial mirtron mimics.…”

“…Most RNAi studies in PD have focused on targeting SNPs in α-syn or LRRK2. In 2006, Sapru et al [22,132] designed shRNAs targeting a SNP in mutant α-syn. Allele-specific silencing was achieved in vitro and in the rat brain.…”

Dopaminergic Dysfunction in Experimental Hepatic Encephalopathy

“…α-synuclein has been shown to form aggregates or insoluble fibrils finally leading to pathological deposits such as those seen in PD, in dementia with Lewy bodies (DLB), and in MSA (Duda et al, 2000;Goedert, 2001;Spillantini et al, 1998;Trojanowski & Lee, 2001;Wakabayashi et al, 2007). Experimental PD models show increased α-synuclein expression and aggregation as well as DAergic neuron degeneration/loss (Cannon & Greenamyre, 2010;Chesselet, 2008;Khodr et al, 2011), accompanied by motor deficit (Khodr et al, 2011).…”