An RNAi strategy for treatment of amyotrophic lateral sclerosis caused by mutant Cu,Zn superoxide dismutase

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts, Worcester, Massachusetts 01605, USA.
Journal of Neurochemistry (Impact Factor: 4.28). 02/2005; 92(2):362-7. DOI: 10.1111/j.1471-4159.2004.02860.x
Source: PubMed


Amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) is a neurodegenerative disease characterized by motor neuron degeneration, paralysis and death. One cause of this disease is mutations in the Cu,Zn superoxide dismutase (SOD1) gene. As mutant SOD1 acquires a toxic property that kills motor neurons, by reducing the mutant protein the disease progression may be slowed or prevented. While mutant SOD1 is toxic, the wild-type SOD1 is indispensable for motor neuron health. Therefore, the ideal therapeutic strategy would be to inhibit selectively the mutant protein expression. Previously we have demonstrated that RNA interference (RNAi) can selectively inhibit some mutant SOD1 expression. However, more than 100 SOD1 mutants can cause ALS and all mutants cannot be inhibited selectively by RNAi. To overcome this obstacle, we have designed a replacement RNAi strategy. Using this strategy, all mutants and wild-type genes are inhibited by RNAi. The wild-type SOD1 function is then replaced by designed wild-type SOD1 genes that are resistant to the RNAi. Here we demonstrate the concept of this strategy.

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    • "Infected cells were further challenged with MPP+ (Sigma) at indicated concentrations for defined times. Cell viability was determined using a 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay according to manufacturer's instruction (Promega) as described previously 25. Statistical significance between experimental groups was analyzed by unpaired t test and p value less than 0.05 was considered of significance. "
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    ABSTRACT: Parkinson's disease primarily results from progressive degeneration of dopaminergic neurons in the substantia nigra. Both neuronal toxicants and genetic factors are suggested to be involved in the disease pathogenesis. The mitochondrial toxicant 1-methyl-4-phenylpyridinium (MPP(+)) shows a highly selective toxicity to dopaminergic neurons. Recent studies indicate that mutation in the vacuolar protein sorting 35 (vps35) gene segregates with Parkinson's disease in some families, but how mutation in the vps35 gene causes dopaminergic cell death is not known. Here, we report that enhanced VPS35 expression protected dopaminergic cells against MPP(+) toxicity and that this neuroprotection was compromised by pathogenic mutation in the gene. A loss of neuroprotective functions contributes to the pathogenesis of VPS35 mutation in Parkinson's disease.
    Preview · Article · Jan 2013 · International journal of biological sciences
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    • "The effective silencing of both alleles of a gene implicated in a disease can be accompanied by the exogenous expression of the normal allele to preserve normal protein function. This so-called replacement strategy was previously tested for SCA6 [39,40], ALS (amyotrophic lateral sclerosis) [41] and RP (retinitis pigmentosa) [42,43]. "
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    Full-text · Article · Mar 2012 · BMC Molecular Biology
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    • "Capitalizing on this capability, it may ultimately permit the exclusive targeting of only the disease-causing mRNAs, while leaving the corresponding wild-type mRNA unaffected to carry out its normal function. Indeed, recent studies have shown that allele-specific silencing by RNAi (ASP-RNAi) is possible and has been applied to disease-causing alleles linked to spinocerebellar ataxia [34], [35], [36], [37], Alzheimer's [38] and Huntington's disease [39], [40], [41], [42], and hereditary amyotrophic lateral sclerosis [40], [43], [44], [45], [46], [47], among others. While initial studies suggested that central positioning of the “mutant recognition site” (MRS) for ASP-RNAi allowed good discrimination, other studies have shown that off-center positioning showed improvements, especially in the case of purine:purine mismatches [40]. "
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    ABSTRACT: Since RNA interference (RNAi) has the potential to discriminate between single nucleotide changes, there is growing interest in the use of RNAi as a promising therapeutical approach to target dominant disease-associated alleles. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been linked to dominantly inherited Parkinson's disease (PD). We focused on three LRRK2 mutations (R1441G/C and the more prevalent G2109S) hoping to identify shRNAs that would both recognize and efficiently silence the mutated alleles preferentially over the wild-type alleles. Using a luciferase-based reporter system, we identified shRNAs that were able to specifically target the R1441G and R1441C alleles with 80% silencing efficiency. The same shRNAs were able to silence specifically mRNAs encoding either partial or full-length mutant LRRK2 fusion proteins, while having a minimal effect on endogenous wild-type LRRK2 expression when transfected in 293FT cells. Shifting of the mutant recognition site (MRS) from position 11 to other sites (4 and 16, within the 19-mer window of our shRNA design) reduced specificity and overall silencing efficiency. Developing an allele-specific RNAi of G2019S was problematic. Placement of the MRS at position 10 resulted in efficient silencing of reporters (75-80%), but failed to discriminate between mutant and wild-type alleles. Shifting of the MRS to positions 4, 5, 15, 16 increased the specificity of the shRNAs, but reduced the overall silencing efficiency. Consistent with previous reports, these data confirm that MRS placement influences both allele-specificity and silencing strength of shRNAs, while further modification to hairpin design or MRS position may lead to the development of effective G2019S shRNAs. In summary, the effective shRNA against LRRK2 R1441 alleles described herein suggests that RNAi-based therapy of inherited Parkinson's disease is a viable approach towards developing effective therapeutic interventions for this serious neurodegenerative disease.
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