Discovery of a new molecular pathway in spinocerebellar ataxia 17

Written by Dr. Sriram Jayabal Edited by Dr. Ray Truant

A potential new pathway for SCA17: gene therapy that in mice restores a critical protein deficit protects brain cells from death in SCA17.

Neurodegenerative ataxias are a group of brain disorders that progressively affect one’s ability to make fine coordinated muscular movements. This makes is difficulty for people with ataxia to walk. Spinocerebellar ataxia type 17 (SCA17) is one such late-onset neurological disease which typically manifests at mid-life. The life expectancy after symptoms first appear is approximately 18-20 years. Besides ataxia, SCA17 can cause a number of other symptoms ranging from dementia (loss of memory), psychiatric disorders, dystonia (uncontrollable contraction of muscles), chorea (unpredictable muscle movements), spasticity (tightened muscles), and epilepsy.

Brain imaging and post-mortem studies have identified that the cerebellum (often referred to as the little brain) is one of the primary brain regions that is affected. That being said, other brain regions such as the cerebrum (cortex or the big brain) and brainstem (distal part of the brain found after the cerebellum) could undergo degeneration. Further, the genetic mutation that leads to SCA17, is a CAG-repeat expansion mutation, similar to several other forms of ataxias. In most other ataxias, where the function of the mutated protein is unknown. However in SCA17, the function of the mutated protein, TATA-box binding protein, is very well understood. Despite this unique advantage, we are yet to completely understand how the mutant gene leads to SCA17. This is why current treatment strategies often focus on treating the symptoms, but not the underlying cause.

person holding laboratory flask
Photo by Chokniti Khongchum on

SCA17 mutation leads to Purkinje cell death

Researchers from China have shed more light on how the mutant gene causes SCA17. TATA-box binding protein is a transcription initiation factor is a protein that turns on the production of RNA from genes. It is widely found across the brain including the cerebellum. TATA-box binding protein controls the amount of protein manufactured from several genes. This raised a very important question: pertinent not only to SCA17 but also more generally to several SCAs – why is that the cerebellar neurons, especially the most sensitive neuron, the Purkinje cells die?

Continue reading “Discovery of a new molecular pathway in spinocerebellar ataxia 17”

Snapshot: The next-generation of CRISPR is prime editing – what you need to know

The CRISPR gene-editing toolbox expanded with the addition of prime editing. Prime editing has astounding potential for both basic biology research and for treating genetic diseases by theoretically correcting ~89% of known disease-causing mutations.

What is prime editing?

Prime editing is coined as a “search-and-replace” editing technique that builds on the “search-and-cut” CRISPR technology. Like CRISPR, prime editing utilizes the Cas9 enzyme targeted to a specific location in the genome by a guide RNA (gRNA). With a few ingenious modifications, including an enzyme called a reverse transcriptase (RT) fused to Cas9, prime editors can be targeted to nearly anywhere in the genome where the RT writes in new DNA letters provided by a template on the gRNA.

graphic drawing of red handled scissors
New gene-editing techniques offer more opportunities for therapy development. Each new discovery makes the techniques more and more accurate. Image courtesy of yourgenome.

 How is prime editing different from CRISPR?

Scientists are excited about prime editing because it has several advantages and overcomes many of the limitations of previous CRISPR systems. CRISPR Cas9, an endonuclease, cuts—like scissors—both DNA strands to inactivate a gene or to insert a new sequence of donor DNA. Unlike CRISPR edits, the prime editing Cas9, a nickase, cuts a single DNA strand and does not rely on the cell’s error-prone repair machinery, thereby minimizing any resulting deleterious scars left on the DNA. It has a broader range of targets because it is not limited by the location of short DNA sequences required for Cas9 binding to DNA. The versatility and flexibility of the system allows for more control to inactivate genes as well as to insert, remove, and change DNA letters, and, combine different edits simultaneously—analogous to a typewriter. Importantly, the edits are precise with relatively infrequent unwanted edits. Initial indications showed fewer off-target edits in the genome, possibly because more steps are required for a successful edit to occur. In some cases, it may be more efficient than CRISPR, depending on the targeted cell type, such as in a non-dividing cell like a neuron in the brain. However, with all these advantages, CRISPR still remains the tool of choice for making large DNA deletions and insertions because the prime editing system is limited by the RT and template RNA length.

How could prime editing help ataxia patients?

Prime editing offers enormous possibility for correcting heritable ataxia mutations accurately and safely. In dominantly inherited SCAs, like SCA1 or SCA2, prime editing could shorten the pathogenic repeat expansion allele to the normal length, or inactivate the pathogenic allele without creating unwanted, deleterious mutations. It also provides researchers with a powerful tool to study disease-causing genes in cells and animal models in new ways to advance our knowledge about the underlying mechanisms in ataxia.

What barriers are there to using prime editing as a treatment?

Prime editing will require rigorous testing in cells and animals before moving into humans in a clinical trial. Optimizing delivery and efficiency in target cells and tissues, and minimizing side-effects will be the key barriers to overcome.

To read the original Nature article describing prime editing, it can be found from the Liu lab here.

If you would like to learn more about Prime Editing, take a look at these news stories by The Broad Institute and Singularity Hub.

Snapshot written by Bryan Simpson and edited by Dr. Hayley McLoughlin.

New Strategy for Reducing Ataxin-1 Levels Shows Promise

Written by Carrie A. Sheeler Edited by Dr. Ronald A.M. Buijsen

RNAi reduces levels of disease-causing Ataxin-1 in SCA1 model mice, easing symptoms of disease when injected both before and after symptom onset.

Lowering the amount of the disease-causing mutant Ataxin-1 protein in affected cells and tissues improves symptoms of disease in spinocerebellar ataxia type 1 (SCA1) mouse models. Like patients with SCA1, mouse models exhibit worsening coordination and degeneration of neurons, beginning in adulthood. Previous work has used genetic manipulation before disease onset (Zu et al 2004). This prevents or delays the onset of disease in SCA1 mouse models. When this is done soon after the onset of symptoms, associated markers of disease are reversed. This suggests that there is a window of time after symptoms start wherein mutant Ataxin-1 can be targeted to improve patient outlook. The 2016 paper by Keiser and colleagues seeks to further study this effect, using RNA interference as a strategy to reduce disease-causing levels of Ataxin-1. As there is no current treatment for Ataxin-1, this is an important step towards assessing possible treatment strategies that could be useful in patients.

female scientist holding a clipboard standing in a laboratory in fornt of a microscope. Books and pictures of neurons line the wall behind her
Cartoon of a scientist reading over results.

Current strategies seek to decrease the amount of Ataxin-1 made in cells by targeting messenger RNA (mRNA)- the blueprints for proteins in a cell- for destruction. RNA interference (RNAi) is one such method which harnesses normal cellular processes to degrade specific mRNAs. In Keiser’s 2016 paper, a modified virus carrying a short sequence of DNA is injected into the brain of a mouse with SCA1. When this virus is injected, the DNA sequence enters the cells of nearby brain regions and stops the production of specific mRNA. In this case, it is Ataxin-1 mRNA that is specifically targeted. As Ataxin-1 mRNA are destroyed, the amount of Ataxin-1 protein made in the cell decreases.

Continue reading “New Strategy for Reducing Ataxin-1 Levels Shows Promise”

Snapshot: What is CRISPR?

A common nuisance for bacteria is the bacteriophage: a virus that uses the internal machinery of a bacteria to replicate its own genetic material. Bacteriophages do this by latching onto bacteria and injecting their DNA into the cell. As the cell grows and divides, the bacteriophage’s hope is that their genetic material is replicated alongside the bacteria’s own genome. Unfortunately for bacteriophages, many bacteria have evolved a method to fight off their attacks. After recognizing a viral infection, the bacteria integrate portions of the injected viral DNA into their own genome. The area where these viral DNA segments end up is known as the CRISPR sequence (short for clustered regularly interspaced short palindromic repeat). The viral DNA segments that were integrated into the CRISPR sequence are then replicated and attached to a bacterial protein called Cas9 (CRISPR-associated protein 9). These CRISPR-Cas9 pairs patrol the cell, acting as the bacteria’s antiviral immune system. If the same viral infection happens again, the DNA in one of the CRISP-Cas9 pairs will match part of the injected viral DNA and bind to it. Once bound, Cas9 cuts the viral DNA, which is then destroyed.

a DNA molelcule that has a fragment cut out of it. Scientific drawing and scribble are faint in the background
Artist’s cartoon of DNA that has been cut by CRISPR. Image courtesy of the NIH.

Recently, scientists have found a way to harness this system for manipulating genes (a process broadly called genetic engineering). By making an artificial CRISPR sequence, attaching that sequence to Cas9, then introducing the man-made CRISPR-Cas9 into a cell, it becomes possible to make a targeted cut in any gene. Making a CRISPR-Cas9 pair that targets one specific gene is as simple as making a CRISPR sequence that matches that gene.

Unlike in bacteria, most organisms repair rather than simply destroy cut DNA. This leaves the targeted genetic sequence available for further manipulation, including the introduction of a short mutation or even the insertion of a whole new DNA sequence. In essence, using the CRISPR-Cas9 system, scientists are now able to edit genes in a simple, targeted way.

CRISPR-Cas9 has become quite popular as a genetic tool in research settings: as of now, the genomes of anything from worms and fruit flies to mice and monkeys have been altered using this technique. While its use in humans is still in its early stages – the first patient treated using CRISPR began therapy earlier this year – is plausible that CRISPR-Cas9 could prove useful in altering the genomes of patients with genetic disorders (like, for instance, the SCAs). For patients, this might sound like a miracle cure. However, it is important to note that several concerns remain as to the ethics of human genetic engineering – the concept of “designer babies” being one of them.

If you’re interested in reading more about the conversation around CRISPR and bioethics, check out the articles by NPR and the National Human Genome Research Institute.

Snapshot written by Logan Morrison and edited by Dr. Maxime W. Rousseaux.

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
Photo of a stethoscope by Negative Space on

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.