Snapshot: What is Gene Therapy?

Gene therapy is using nucleic acids to treat a genetic disorder.  These nucleic acids can be designed in a variety of ways to achieve the same therapeutic outcome. Gene therapy tools can be used to correct a mutant gene by one of three ways:

  1. Expressing a healthy copy of a gene
  2. Silencing or inactivating the mutant gene transcript
  3. Using genome editing tools to repair or turn-off the mutated gene.
computer desk laptop stethoscope
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How is gene therapy used?

Monogenic disorders, like some spinocerebellar ataxias (SCAs), are excellent targets for gene therapy approaches. Gene therapies are currently being used throughout ataxia research for studying disease mechanisms and for preclinical therapeutic application.

Overview of how gene therapy works. First, Package the healthy gene, RNAi, or gene editing tools into the AAV (can also deliver as naked DNA or in a nanoparticle). Second, Inject the packaged AAV into the tissue of interest. Third, AAV will enter the cell and release the genetic material. The cell will become healthy by either 1) expressing the normal gene, 2) repressing the mutant RNA, or by 3) correcting the mutant gene.
Overview of gene therapy, designed by Stephanie Coffin using Biorender.

One gene therapy approach for rescuing SCA1 phenotypes involves overexpressing a healthy gene, ataxin-1-like, which competes with the mutant ATXN1 protein for complex formation. This work, conducted by Keiser and colleagues in 2016, showed phenotypic rescue in a mouse model of SCA1.

There are two common technologies for silencing or inactivating disease genes: RNA interference (RNAi) or antisense oligonucleotides (ASOs). RNAi strategies utilize small RNA molecules to knock down the expression of target mutant RNA transcripts, while ASOs are DNA molecules used to knock down or correct mutant RNA transcripts. Both therapeutic approaches are being pursued in SCAs. For example, Carmo and colleagues in 2013 showed that using RNAi against the SCA3 disease gene, ATXN3, could longitudinally decrease mutant ATXN3 levels. See the SCAsource snapshot on ASOs for further information about their use in SCAs.

The most common genome editing tool is the CRISPR/Cas9 system, which uses an RNA guide to direct the Cas9 nuclease to the region of the genome to be edited. One can then knockout that gene or correct the mutant gene. It is early days for this technology as a potential therapeutic option due to the challenges of delivery and the risk of off-target editing.

How is gene therapy delivered?

One of the most difficult aspects of gene therapy is how to deliver these various molecules to the cells of interest. One of the most common delivery methods is through viral delivery.  The “drug” nucleic acid is transferred into the disease cells by a vector, which is a virus that has been modified to remove viral components. The most common viral vectors for gene therapies currently are adeno-associated viruses (AAVs). Other delivery methods include non-viral vectors such as naked DNA and nanoparticles.

How long-lasting is gene therapy?

Viral delivery of gene therapy products provides a longitudinal expression of the nucleic acid, while naked DNA and nanoparticles express the nucleic acid drug transiently, thus typically requiring ongoing treatment.

If you would like to learn more about gene therapy, take a look at these resources by the National Institutes of Health and KidsHealth.

Snapshot written by Stephanie Coffin and edited by Dr.Hayley McLoughlin.

Snapshot: What is an antisense oligonucleotide (ASO/AON)?

Antisense Oligonucleotides (also known as ASOs or AONs) are small molecules that can be used to prevent or alter the production of proteins. Proteins are the workforce of the cell, taking care of most cellular processes. They are generally made in a two-step process: first, a specific protein-coding gene is converted into an instruction file, called the messenger RNA (mRNA). The mRNA carries the information from that gene to the compartment of the cell that builds proteins. There, the mRNA’s information then gets converted into the protein. ASOs are short single stranded pieces of DNA that match the complementary sequence of a specific mRNA. Based on the type of chemical modifications, the ASO can have two different effects on the mRNA. Some modifications of ASOs trigger the destruction of the mRNA. This will result in the loss of the corresponding protein. Other modifications can mask only certain parts of the mRNA leading to a modified version of the protein.

diagram showing how ASOs can bind or snip mRNA to create alternative forms of protein or prevent protein synthesis
How ASOs work in the human body. Image by Larissa Nitschke, Created with BioRender.

The majority of Spinocerebellar Ataxias (SCAs) are caused by the accumulation of toxic proteins in certain regions of the brain. The primary goal of ASO treatments for SCAs is therefore to prevent the production of the toxic protein altogether. One example is work from Dr. Harry Orr’s group at the University of Minnesota. His lab studies Spinocerebellar Ataxia Type 1 (SCA1), which is caused by the toxic accumulation of the Atxn1 protein. Injections of ASOs into a SCA1 animal model decreased Atxn1 levels and rescued the SCA1 motor incoordination symptoms. Another way of using ASOs as treatment for SCAs is the modification of the mRNAs information to produce a modified version of the protein. This approach has been tested in Spinocerebellar Ataxia Type 3 (SCA3), in which an expansion in the Atxn3 gene renders the Atxn3 protein toxic. The van Roon-Mom group from the Netherlands, for instance, used ASOs to only remove the expansion from Atxn3 while leaving the remaining protein structure and function intact.

Both studies as well as other studies performed for additional SCAs are highlighting the potential use of ASOs as therapeutics for SCAs. While ASO research for SCA is mostly in the pre-clinical phase, ASO treatment for other diseases, including Duchenne muscular dystrophy and spinal muscular atrophy, have already gained approval by the US Food and Drug Administration (FDA). Further clinical trials will need to be performed to measure the therapeutic benefit of ASOs in SCA patients.

If you would like to learn more about antisense oligonucelotides, take a look at this article in HDBuzz about ASOs in development for Huntington’s Disease.

Snapshot written by Larissa Nitschke edited by Dr. Hayley McLoughlin



Approaching the age of clinical therapy for spinocerebellar ataxia type 1

Written by Dr. Marija Cvetanovic Edited by Dr. Maxime W. Rousseaux

New research (published Nov. 2018) reveals promising potential genetic therapy for SCA1.

A research team comprised of scientists from academia and industry have tested a new treatment for Spinocerebellar ataxia type 1 (SCA1), bringing disease-modifying therapy one step closer to the clinic. SCA1 is a dominantly-inherited ataxia that is currently untreatable. Symptoms of the disease include progressive loss of balance, slurring of speech, difficulties with swallowing and coughing, mild cognitive impairments, and depression. With a life expectancy after diagnosis of only 10-15 years, SCA1 is one of the fastest-progressing SCAs: after symptoms first appear, patients typically have just over a decade before these symptoms become so severe that they cause death (often due to respiratory failure). In 1993, collaborative efforts from the laboratories of Drs. Harry T. Orr and Huda Y. Zoghbi discovered that SCA1 is caused by the expansion of a CAG repeat somewhere in a patient’s DNA. CAG repeats cause a polyglutamine expansion in the protein that the mutated gene encodes; in this case, the group later identified that this had occurred in Ataxin-1 (ATXN1), the gene that encodes the ATXN1 protein. The SCA1 mouse models that Drs. Orr and Zoghbi generated (and graciously shared with the scientific community) have allowed for significant advances in the understanding of SCA1 pathogenesis over the years. Now, they provide preclinical evidence of a promising therapy to alter the progressive motor deficits and fatal outcome of SCA1.

stethoscope on top of laptop
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A novel therapeutic approach for the treatment of SCA3

Written by Larissa Nitschke Edited by Dr. Gülin Öz

Researchers in the Netherlands uncover a new way to treat SCA3

Upon receiving a conclusive diagnosis of Spinocerebellar Ataxia (SCA), hundreds of questions can appear in a patient’s mind: What is Spinocerebellar Ataxia? Why am I affected? How will my symptoms progress? What is the ultimate prognosis? Thankfully, years of research have enabled us to answer many of these questions for patients affected by Spinocerebellar Ataxia Type 3 (SCA3), also known as Machado-Joseph Disease. Still, the most important question a patient could ask – How can I be healthy again? – has remained unanswered.

SCA3 is the most common form of Spinocerebellar Ataxia worldwide. It is passed down from generation to generation in affected families. Initial symptoms typically appear around midlife, but cases of much earlier and much later onset have been reported. At first, problems with movement coordination are the most noticeable, leading to an increase in stumbles and falls. At later stages, speech difficulties, muscle stiffness, and sleeping problems appear, leaving the patient fatigued during the day. The symptoms worsen over the course of 10 to 20 years, at which point affected individuals typically succumb to the disease. As with other SCAs, current options for SCA3 treatment are mainly limited to symptom management rather than treating the direct cause of the disease.

Artist's representation of DNA
Artist’s representation of DNA. Photo from Pixabay.

The genetic cause of SCA3 is the presence of excess copies of the DNA building blocks cytosine (C), adenine (A), and guanine (G) in the Ataxin-3 gene (Atxn3). Scientists refer to this type of mutation as an expansion of a triplet repeat, since the C, A, and G copies appear as sets of back-to-back CAGs. Because the CAG triplet is responsible for coding the amino acid glutamine (Gln or Q) in the Ataxin-3 protein, the repeat expansion results in an elongated glutamine (polyQ) tract. This faulty protein accumulates in cells and causes toxicity in specific regions of the brain. Since the 1994 discovery that SCA3 is caused by a polyQ expansion in Atxn3, scientists and physicians all over the world have been humbled by the question of how to help patients affected with SCA3. One specific angle of research has focused on the removal of the toxic protein altogether. However, one downside of this approach is that it would also cause the loss of normal Atxn3 function in patients. Atxn3 is critical for the degradation of unwanted proteins, which is necessary for the healthy functioning of all our body’s cells. It normally binds to little marks on proteins called ubiquitin chains (which tag proteins for removal), then cleaves these chains to facilitate the entry of proteins into the cell’s destruction machinery. Since treatment will need to be sustained over the span of a patient’s lifetime, the complete removal of Atxn3 might be harmful.

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ASOs clear toxic protein from cells, reducing ataxia in SCA2 mice

Written by Anna Cook and Dr. Alanna Watt Edited by Dr. Vitaliy V. Bondar

Scientists uncover a promising therapeutic avenue to treat spinocerebellar ataxia type 2 (SCA2).

Spinocerebellar ataxia type 2 (SCA2) is a progressive ataxia caused by a mutation in the ATXN2 gene. This mutation causes a tract of the amino acid glutamine in the ataxin 2 protein to expand, making it toxic to cells. This type of mutation – known as a polyglutamine expansion – is common to several neurodegenerative diseases, including Huntington’s Disease and several forms of ataxia. One treatment strategy that has been devised for polyglutamine diseases such as SCA2 is to remove the toxic protein from cells. And, in their tour de force SCA2 paper from 20171, this is precisely what Scoles and colleagues attempted to do. Removing protein levels is a particularly promising strategy for SCA2, since previous research from the authors of this paper has shown that a complete loss of healthy ataxin 2 protein in cells does not cause any major detectable behavioural consequences in mice2.

Removing a toxic protein from a cell is not a simple task; in fact, it has only been done a handful of times in models of neurodegeneration. One way to eliminate a protein in neurons is to cause the RNA that encodes it to be degraded before it can make the protein. Through a collaboration with a company that specializes in this approach — Ionis Pharmaceuticals — the authors created their own short RNA molecules that matched the sequence and therefore bound to regions in the specific RNA that encodes the protein ataxin 2. These small molecules are known as anti-sense oligonucleotides (ASOs), and once they bind to their partner, they recruit the cell’s waste system to degrade the RNA. Currently, ASO therapy is one of the most promising methods researchers have developed to eliminate toxic proteins for a wide range of degenerative diseases.

blue stethoscope next to laptop computer
Image of stethoscope next to a computer. Photo by Negative Space on

After designing many of these molecules, the authors screened 152 different ASOs to determine which were most effective at lowering levels of the toxic protein. ASOs were applied to skin cells that had been donated by SCA2 patients, and levels of mutated ataxin 2 protein were measured. By picking out the designs that caused the greatest decrease in ataxin 2 levels, the authors narrowed down the original group of potential ASOs to give a shortlist of promising candidates. The authors then chose one ASO (ASO7) to test in mouse models of SCA2.

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