Therapeutic Gene Silencing Delivered by a Chemically Modified Small Interfering RNA against Mutant SOD1 Slows Amyotrophic Lateral Sclerosis Progression

Wang, Hongyan; Ghosh, Animesh; Baigude, Huricha; Yang, Chyun-Yu; Qiu, Linghua; Xia, Xu‐Gang; Zhou, Hongxia; Rana, Tariq M.; Xu, Zuoshang

doi:10.1074/jbc.m800834200

Cited by 58 publications

(52 citation statements)

References 41 publications

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“…6,11,21 However, only a few studies have assessed silencing in patient-derived cells, which exhibit physiologically relevant levels of endogenous expression. 3,[22][23][24][25] We sought to address whether polyQ patient-derived fibroblasts might be a useful model for assessing therapeutic strategies, such as comparing allele-specific and non-allele-specific silencing approaches in genetically accurate cells.…”

Section: Discussionmentioning

confidence: 99%

Allele-specific silencing of mutant Ataxin-7 in SCA7 patient-derived fibroblasts

et al. 2014

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Polyglutamine (polyQ) disorders are inherited neurodegenerative conditions defined by a common pathogenic CAG repeat expansion leading to a toxic gain-of-function of the mutant protein. Consequences of this toxicity include activation of heatshock proteins (HSPs), impairment of the ubiquitin-proteasome pathway and transcriptional dysregulation. Several studies in animal models have shown that reducing levels of toxic protein using small RNAs would be an ideal therapeutic approach for such disorders, including spinocerebellar ataxia-7 (SCA7). However, testing such RNA interference (RNAi) effectors in genetically appropriate patient cell lines with a disease-relevant phenotype has yet to be explored. Here, we have used primary adult dermal fibroblasts from SCA7 patients and controls to assess the endogenous allele-specific silencing of ataxin-7 by two distinct siRNAs. We further identified altered expression of two disease-relevant transcripts in SCA7 patient cells: a twofold increase in levels of the HSP DNAJA1 and a twofold decrease in levels of the de-ubiquitinating enzyme, UCHL1. After siRNA treatment, the expression of both genes was restored towards normal levels. To our knowledge, this is the first time that allelespecific silencing of mutant ataxin-7, targeting a common SNP, has been demonstrated in patient cells. These findings highlight the advantage of an allele-specific RNAi-based therapeutic approach, and indicate the value of primary patient-derived cells as useful models for mechanistic studies and for measuring efficacy of RNAi effectors on a patient-to-patient basis in the polyQ diseases.

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Section: Discussionmentioning

confidence: 99%

Allele-specific silencing of mutant Ataxin-7 in SCA7 patient-derived fibroblasts

et al. 2014

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“…The proposed protocol is a guanidinium salt-free, phenol-based method. It is evident from the researches of Wang et al (2008), the presence of guanidinium salt stimulates RNA dissociation from the non-protein complex and that will further results in inhibition of successful RNA isolation. Phenol act as a strong protein denaturant and RNase inhibitor.…”

Section: Discussionmentioning

confidence: 99%

“…The phenolic compounds readily get oxidised to form quinones. These quinones can bind easily with nucleic acids and act as a barrier for good quality RNA isolation (Wang et al 2008).…”

Section: Introductionmentioning

confidence: 99%

“…The phenolic compounds readily get oxidised to form quinones. These quinones can bind easily with nucleic acids and act as a barrier for good quality RNA isolation (Wang et al 2008).Several RNA isolation protocols for medicinal plants has been developed or modified and certain commercial kits were also introduced (John 1992, MacKenzie et al 1997, Sabir 2012. However, these protocols were found to be incompatible for RNA isolation from majority of medicinal plant species with higher…”

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Simple and efficient method for functional RNA extraction from tropical medicinal plants rich in secondary metabolites

George¹

2018

Trop. Plant Res.

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Isolation of high-quality functional RNA is prerequisite to facilitate any study related to gene expression and also for downstream applications such as reverse transcription-polymerase chain reaction (RT-PCR), suppression subtractive hybridization (SSH) library construction, differential display (DD), real-time PCR and northern hybridization of medicinal plants. Tropical medicinal plants are rich in polysaccharides, polyphenolics and secondary metabolites that are reported to interfere with the successful RNA isolation. Conventional approaches for the extraction of functional RNA are often time-consuming and need expensive reagents. Moreover, these methods can yield only poor quality and quantity of functional RNA. In our laboratory, we have aimed at establishing a simple and efficient functional RNA extraction procedure. For this, a sodium dodecyl sulfate (SDS) based protocol free of guanidine isothiocyanate was adopted. The total extracted RNA remains in the upper aqueous phase, at acidic conditions leaving DNA and proteins in the lower organic phase. This principle is used in this protocol and the modifications made in the SDS-acid phenol RNA extraction protocol were found to be helpful for extracting RNA from tissues containing large quantities of secondary metabolites. This method can be completed within 3 hours with purity ranges from 1.8-2.0 as confirmed by A 260/280 spectrophotometric readings. The above-described RNA extraction protocol works well with all the tissues examined so far, where standard RNA isolation methods failed. INTRODUCTIONTropical medicinal plants have been largely exploited for therapeutic purposes by present-day researchers. Effective conservation and sustainable utilization of plant biodiversity is indispensable for meeting the present and future requirements of medicinal plants. These medicinal plants are rich sources of alkaloids, polyphenols, polysaccharides, secondary metabolites, terpenoids and these phyto compounds have dragged the attention of contemporary researchers. Production of valuable therapeutic compounds from medicinal plants could be made possible through metabolic engineering and recombinant DNA technology (Titanji et al. 2007). The application of modern molecular biology techniques may help in better understanding and conservation of medicinal plants. These tools can unravel the various bioactive compounds with therapeutic significance at the molecular level. Good quality functional RNA extraction is the most important step in gene expression analysis studies (Ghangal et al. 2009). However, RNA extraction from tissues having higher polyphenols, polysaccharides and secondary metabolite contents became a difficult task. The phenolic compounds readily get oxidised to form quinones. These quinones can bind easily with nucleic acids and act as a barrier for good quality RNA isolation (Wang et al. 2008).Several RNA isolation protocols for medicinal plants has been developed or modified and certain commercial kits were also introduced (John 1992, MacKenzie e...

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“…We found that scAAV5-GABRA4-TREK-M pre injected rats had significantly less fig 8F). However, no reduction was observed in FJC+ cells in the dentate gyrus, where neuronal death predominantly occurs in the hilus as others have observed previously (Wang, Ghosh et al 2008;Sun, Mtchedlishvili et al 2007). In order to quantify the number of dead cells which is represented by green colored FluoroJade -C stained neurons, we calculated the Fluoro-Jade C positive neurons in each brain slice.…”

Section: Trek-m Protected Against Neuronal Deathmentioning

confidence: 62%

Silencing Hyper-Excitable Neurons as a Treatment for Status Epilepticus and Temporal Lobe Epilepsy.

Dey¹

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Therapeutic Gene Silencing Delivered by a Chemically Modified Small Interfering RNA against Mutant SOD1 Slows Amyotrophic Lateral Sclerosis Progression

Cited by 58 publications

References 41 publications

Allele-specific silencing of mutant Ataxin-7 in SCA7 patient-derived fibroblasts

Allele-specific silencing of mutant Ataxin-7 in SCA7 patient-derived fibroblasts

Simple and efficient method for functional RNA extraction from tropical medicinal plants rich in secondary metabolites

Silencing Hyper-Excitable Neurons as a Treatment for Status Epilepticus and Temporal Lobe Epilepsy.

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