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: 4.24). 02/2005; 92(2):362-7. DOI: 10.1111/j.1471-4159.2004.02860.x
Source: PubMed

ABSTRACT 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.

Download full-text


Available from: Zuoshang xu, Jun 11, 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: RNA interference (RNAi) is a potential cure for amyotrophic lateral sclerosis (ALS) caused by dominant, gain-of-function superoxide dismutase 1 (SOD1) mutations. The success of such therapy relies on the functional small interfering RNAs (siRNAs) that can effectively deliver RNAi. This study aimed to design the functional siRNAs targeting ALS-associated mutant alleles. A modified dual luciferase system containing human SOD1 mRNA target was established to quantify siRNA efficacy. Coupled with validated siRNAs identified in the literature, we analyzed the rationale of siRNA design and subsequently developed an asymmetry rule-based strategy for designing siRNA. We then further tested the effectiveness of this design strategy in converting a naturally symmetric siRNA into functional siRNAs with favorable asymmetry for gene silencing of SOD1 alleles. The efficacies of siRNAs could vary tremendously by one base-pair position change. Functional siRNAs could target the whole span of SOD1 mRNA coding sequence as well as non-coding region. While there is no distinguishable pattern of the distribution of nucleobases in these validated siRNAs, the high percent of GC count at the last two positions of siRNAs (P18 and P19) indicated a strong effect of asymmetry rule. Introducing a mismatch at position 1 of the 5' of antisense strand of siRNA successfully converted the inactive siRNA into functional siRNAs that silence SOD1 with desired efficacy. Asymmetry rule-based strategy that incorporates a mismatch into siRNA most consistently enhances RNAi efficacy and guarantees producing functional siRNAs that successfully silence ALS-associated SOD1 mutant alleles regardless target positions. This strategy could also be useful to design siRNAs for silencing other disease-associated dominant, gain-of-function mutant genes.
    Chinese medical journal 01/2011; 124(1):106-10. · 1.02 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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.
    International journal of biological sciences 01/2013; 9(2):149-55. DOI:10.7150/ijbs.5617 · 4.37 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Transgenic RNAi, an alternative to the gene knockout approach, can induce hypomorphic phenotypes that resemble those of the gene knockout in mice. Conditional transgenic RNAi is an attractive choice of method for reverse genetics in vivo because it can achieve temporal and spatial silencing of targeted genes. Pol III promoters such as U6 are widely used to drive the expression of RNAi transgenes in animals. Tested in transgenic mice, a Cre-loxP inducible U6 promoter drove the broad expression of an shRNA against the Pink1 gene whose loss-of-functional mutations cause one form of familial Parkinson's disease. The expression of the shRNA was tightly regulated and, when induced, silenced the Pink1 gene product by more than 95% in mouse brain. However, these mice did not develop dopaminergic neurodegeneration, suggesting that silencing of the Pink1 gene expression from embryo in mice is insufficient to cause similar biochemical or morphological changes that are observed in Parkinson's disease. The results demonstrate that silencing of the PINK1 gene does not induce a reliable mouse model for Parkinson's disease, but that technically the inducible U6 promoter is useful for conditional RNAi in vivo.
    International journal of biological sciences 03/2007; 3(4):242-50. · 4.37 Impact Factor