RNAi for neurological diseases

Published on August 5, 2014   48 min

Other Talks in the Series: Gene Transfer and Gene Therapy

Other Talks in the Series: RNA Interference

RNAi for Neurological Diseases. My name is Beverly Davidson. I work at the Center for Cell and Molecular Therapy at the Children's Hospital of Philadelphia.
I'm going to talk today about RNAi interference for repeat expansion diseases. Shown is a schematic of a gene indicating a disease repeat sequence in the location of the repeat within the gene. For example, all but one CAG repeat, SCA12 is in the protein coding region. These, therefore, encode polyglutamine in the disease containing proteins. Other repeats, for example the GAA in Friereich's Ataxia, is in a non-coding region, in this instance, in the intronic region. There are examples of other repeats, for example CTG repeats in three prime UTRs in mitotic dystrophy, also known as DM1. And also CGG repeats in the Fragile X locus. These occur in the five prime UTR. Purposes of today's talk, I'm going to focus on the repeat expansion diseases that are due to polyglutamine expansion, or CAG repeat expansion, in the genes that, when mutated, caused the diseases known as spinocerebellar ataxia or SCA type one, type two, type six, type seven, or Huntington's disease, denoted here as HD. I will only be presenting data on SCA1 and Huntington's disease today, although in my laboratory work on all of those that are highlighted below.
As I mentioned, I'm going to be talking about the polyglutamine repeat expansion diseases that are due to an expanded repeat within the coding region of the disease protein. This cartoon below depicts the expansion of a normal containing CAG repeat. In the diseases I'm talking about today, Huntington's and spinocerebellar ataxia, the repeat ranges in a normal allele, generally between 17 and 30 repeat lengths. In disease alleles, this repeat length is expanded up into the 35 and greater range.
Huntington's disease was first described by Doctor George Huntington, a physician on Long Island. Dr. Huntington noted several peculiarities on this disease. He noted the dominant heritability, the fact that most individuals showed adult onset. He described the chorea, which is defined as the involuntary dance-like movements, the drastic personality changes, insanity, and suicide. Inevitably, Huntington's disease leads to death. In 1983, a mutation underlying Huntington's disease was localized to the short arm of chromosome four, depicted by the red bar on the top of the picture of chromosome four on the right. Another 10 years passed before the Huntington's disease gene was identified. Originally called interesting transcript 15, it was renamed Huntington and gives rise to the Huntington protein. As I said before, the mutation in Huntington's disease is a polyglutamine repeat expansion within exon one of Huntington.
Here we are looking at sections from normal humans or from individuals that have Huntington's disease. One of the regions of the brain that degenerates in Huntington's disease is known as the basal ganglia. And that's identified here by the light green arrow. You can see as we progress from a normal brain on the left panel to Huntington's disease patients with progressive disease from the second to the fourth panels that there's extensive cell loss and atrophy. Also noted and highlighted by the red arrows is the fact that Huntington's brains also undergo significant cortical atrophy.
Mutant Huntington causes many problems in cells that express the protein, and particularly in cells in the brain. It induces trafficking defects, excitotoxicity of neurons, mitochondrial dysfunction in multiple cell types, oxidative damage, transcriptional dis-regulation, and others. Only a partial listing of the problems that Huntington causes with a polyglutamine repeat expansion are listed here. Each of these downstream effects of the mutant protein are targets for therapeutic development. We reasoned was that RNA interference, abbreviated as RNAi, could take out the inducing mutant cause of disease and alleviate all downstream effects.
What is RNA interference? This slide is a cartoon adapted from the reference depicted on the right of the cartoon. In short, primary mRNA transcripts, which encode normally expressed non coding RNAs, is known as microRNAs, are expressed in the nucleus and processed by an enzyme complex that includes a protein called drosha. This processes these hairpin like structures into shorter hairpin like structures known as an micro RNA precursor molecule. These precursors are exported into the cytoplasm, generally in a exportin-5 dependant manner, and are further processed in the nucleus and incorporated into the dicer complex known as DCR1. These trimmed inhibitory RNAs can then go through either a mRNA cleavage pathway or a translational repression pathway, depending on the complementarity between the active strand of this inhibitory RNA and its target transcript. We can intercede in the pathway in several ways in the laboratory, either by expressing shRNAs which mimic MIRNA precursors, or by transfecting cells with siRNAs, which mimic those longer double strand structures without a hair pin. And you can see from this picture where they would enter into the normally occurring microRNA processing pathway.

RNAi for neurological diseases

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