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.

Snapshot: How does CAG tract length affect ataxia symptom onset?

The instructions our bodies need to grow and function are contained in our genes. These instructions are made up of tiny structures called nucleobases. There are four types of nucleobases in DNA: adenine (A), cytosine (C), guanine (G), thymine (T). By putting these four nucleobases in different orders and patterns, this writes the instructions for our body.

artists drawing of a blue DNA molecule
A cartoon strand of DNA. Image by PublicDomainPictures from Pixabay

Some of the genes contain long sections of repeating ‘CAG” instructions, called CAG tracts. Everyone has repeating CAG tracts in these genes, but once they are over a certain length they can lead to disease. Some ataxias are caused by this type of mutation, including SCA1, SCA2, SCA3, SCA6, SCA7 and SCA17. These are often called polyglutamine expansion disorders. This is because “CAG” gives the body instructions to make the amino acid glutamine. You can read more about what is polyglutamine expansion in our past Snapshot about that subject.

For each disorder caused by a CAG expansion mutation, the number of times the CAG is repeated in a particular gene will determine whether someone will develop the disease. Repeat lengths under this number will not cause symptoms and repeat lengths over the threshold will usually lead to ataxia. When someone undergoes genetic testing for ataxia, doctors will be able to tell them the length of these CAG tracts and whether they have a CAG repeat number in one of these genes that is over the threshold. This table gives a summary of different CAG expansion mutations that can lead to ataxia and how the length of the repeat affects age of onset.

Affected Gene Normal
Repeat Size
Disease
Repeat Size
SCA1ATXN16-4439-88
SCA2ATXN215-3136-77
SCA3ATXN312-4055-86
SCA6CACNA1A 4-1821-33
SCA7ATXN74-3537-306
SCA12PPP2R2B4-3266-78
SCA17 TBP25-4246-63

For SCA1, SCA2, SCA3, SCA6, and SCA7; longer CAG tracts are associated with earlier onset.

For SCA12, it is hard to predict the age of onset based on repeat length as SCA12 is so rare. Some individuals with long repeats don’t develop ataxia. One study found that longer CAG tract lengths are associated with earlier onset but that it does not affect the severity of symptoms.

For SCA17, Longer CAG tracts have separately been associated with an earlier age of onset and more severe cerebellar atrophy.

In general, people with longer repeat lengths in ataxia genes are likely to present with ataxia symptoms earlier in life. However, it is important to remember that there are many other factors involved. Other genes may have mutations that either worsen the progression of ataxia or protect against more severe symptoms. Therefore, in individual people, the length of the repeat is not always enough information to determine when that person will start showing symptoms, or how severe these symptoms will be.

If you would like more information about the genetic causes of SCAs, including information about genetic testing and what CAG repeat length might mean, take a look at these resources by the National Ataxia Foundation.

Snapshot written by Anna Cook and edited by Larissa Nitschke.

Continue reading “Snapshot: How does CAG tract length affect ataxia symptom onset?”

Snapshot: What Does Success Mean in Clinical Trials with Antisense Oligonucleotides (ASO)?

Research is rapidly moving from the bench to the bedside to treat neurological inherited disorders of all types, including spinocerebellar ataxias. SCAsource has previously gone over the science behind ASO therapy. These diseases share a common theory that the DNA mutation leads to the formation of an altered protein that is toxic. ASO therapy is meant to stop the formation of the toxic protein by “shooting the messenger”.

What is involved in these clinical trials?

To see what might happen in ataxia trials, let’s look at ASO trials happening right now in related polyglutamine diseases. In Huntington’s disease (HD), there are two programs that are currently in clinical trials. Regulatory authorities view ASOs as drugs and require that the product be shown to be both safe and effective in patients.

ASOs cannot be given as pills and they are currently injected into the spinal fluid. This is called intrathecal administration to get the drug directly in the fluid space where it can circulate back to the brain. Patients in phase 1 studies in HD are asked to have up to 7 injections and one phase 3 program requires injections every second month for 2 years. This involves a large commitment to the study and is asking a lot from patients and their families.

The only published phase 1 double-blind, placebo-controlled study in HD (Tabrizi et al., New England Journal of Medicine, 2019) has identified that a series of 4 injections were safe. They measured changes of the “bad” protein in the spinal fluid as a proof of concept that ASOs could lower protein levels. The good news was that they found that there was a dose-related reduction in this protein of about 40%. Patients from this study were offered “open label” monthly injections and this has shown a 60% reduction in the abnormal protein according to a recent presentation. Open label extensions are when patients can continue taking a drug after the formal time of the clinical trial is over.

medical doctor in blue scrubs and a white lab coat holding a stethoscope. They are off to one side, so only have their body can be seen, not inclduing their face.
What will ataxia clinical trials involving ASOs look like in the future? What will success look like?

So, what does success mean?

The phase 3 studies that are currently ongoing in HD are designed to see if there is a slowing of disease progression. This is being measured by assessing motor, cognitive and behavioral symptom change over time. Changes occur slowly in HD and SCA. Therefore, large numbers of patients are required over a relatively long study time.

The bottom line is that a successful study that shows slowing disease progression is likely to mean that the patients may not experience any obvious improvement while receiving the treatment and that they will continue to have progressive symptoms over time. Hopefully, this will be at a slower rate compared to the placebo group. Since there are no treatments available for SCA or HD, this will be welcome. It is by no means considered to be a cure or likely to stop the progression. True cures in medicine are rare, where a cure is defined as a drug ending disease.

Graphs of symptoms vs time. The "typical progression" line has more symptoms more quickly. The "delayed progression after potential treatment" line has fewer symptoms, but still increases over time.
Graph explaining how a potential ASO treatment might work in the future. Although it might not make symptoms go away completely, it could reduce how severe symptoms are, the number of symptoms, and/or delay when symptoms first appear. Illustration by Celeste Suart.

In the HD research community, we are asking questions that include:

  1. Is it a good idea to reduce the good protein that is part of our normal brain chemistry? In the current phase 3 study, the ASO reduces both the “good” and the “bad” HD protein. Another program in phase 1 uses an ASO that only reduces the “bad” protein.
  2. When is the best time to use ASO therapy? Since these conditions are associated with nerve cell damage and loss, it makes sense to use these types of therapy very early, even before damage occurs. This will mean that patients with moderate or advanced symptoms may not be good candidates for ASO therapy.
  3. Should we consider treatment in people who have had predictive genetic testing before symptoms start? This is being actively discussed but it is too early to consider this. We have to show that ASOs are safe and effective in symptomatic patients. We need to have good measures to determine if treatments are working. Regulatory authorities have required evidence that treatments have a positive effect on patients lives. This may be difficult to show in a short study. We must consider that it takes patients decades to get these diseases: slowing or stopping this could take just as long.

We can only figure out the answers to these questions in clinical trials. The goals of these trials are to improve people’s quality of life. To do this we need information from real people with these diseases, and not just models of disease. This is a process that will take time but will tell us which approach has the most promise and is worth pursuing faster. Thus, the patients and families at this point are just as important as the researchers in lab coats working together to treat these diseases.

If you would like to learn more about clinical trials, take a look at this resource by the FDA or our previous Snapshot on the subject.

Snapshot written by Dr. Mark Guttman and edited by Dr. Ray Truant.

Snapshot: What is recessive ataxia?

What is a recessive disorder?

A recessive disorder is one that has a specific disease mechanism. For a recessive disorder to occur, both copies of the causative gene must be mutated for a patient to show symptoms.  Ataxias that follow this disease mechanism are known as recessive ataxia. However, having a mutation in only one copy of the gene does not lead to a disorder. As people with only one mutated copy of the gene can pass on the defective gene, these people are known as an unaffected carrier. Recessive ataxias range in symptoms and severity but are linked by their disease mechanism. While none of the Spinocerebellar Ataxias (SCAs) are recessive, there are many types of recessive ataxias, including Autosomal Recessive Cerebellar Ataxia Type 1 and 2 (ARCA1 and ARCA2), Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS), Friedreich’s Ataxia, and Ataxia Telangiectasia. For example, Friedreich’s Ataxia is caused by a trinucleotide repeat expansion in the frataxin (FXN) gene. People with only one expanded copy of the FXN gene do not show any symptoms, while people with two expanded copies of the FXN gene are affected by Friedreich’s Ataxia.

How are recessive ataxias inherited?

For every gene in our body, we have two copies, one that is inherited from our mother and one from our father. Both parents of an affected individual have to have at least one copy of the mutation for a child to be born with a recessive disorder. If both parents are unaffected carriers, each child will have a 1 in 4 chance of getting the disorder.

For a patient affected with a recessive ataxia, the chances of having a child affected by the same disorder are low. For a patient to pass on the disease, their spouse must have at least one mutated copy of the causative gene. In the case where a patient’s spouse is a carrier, children have an equal chance of being an unaffected carrier or being affected by the disease. However, carrier rates for ataxias are low in the population, which makes it unlikely that a patient’s spouse is also a carrier for the ataxia mutation.

Map showing the statistical chance of two unnafected carrier parents passing on a mutated gene (25% unaffected child, 50% carrier child, 25% affected child) or an affected parrent and unaffected carrier (50% carrier child, 50% affected child)
How recessive disorders are inherited. Image by Eder Xhako, created with BioRender

How can a patient prevent passing on a recessive disorder to their children?

Generally, when a patient with recessive ataxia passes on the disorder to their children, their spouse is an unaffected carrier. If you are a patient with a form of recessive ataxia and are thinking about having children, your spouse can undergo carrier testing to find out if they are a carrier for the same recessive ataxia. This will determine the likelihood that the recessive ataxia is passed on to your children. If it is determined that the spouse is a carrier, options like IVF with embryo screening can help patients prevent passing on recessive ataxia to their children.

If you would like to know more trinucleotide repeat expansions, you can look at our past Snapshot on Polyglutamine Expansion.

If you would like to learn more about carrier and embryo screening, take a look at these resources by the American College of Obstetricians & Gynecologists and Integrated Genetics.

Snapshot written by Eder Xhako and edited by Larissa Nitschke.

Snapshot: What is Magnetic Resonance Imaging (MRI)?

What is it?

Magnetic resonance imaging (MRI) is a type of technology used to take detailed pictures of the body. It is commonly used to detect abnormalities in the body, diagnose diseases, and to regularly monitor patients who are undergoing treatments. It can generate three-dimensional images of non-bony tissues, such as the brain. MRI procedures are non-invasive, require minimal preparation, and are not associated with health risks, as it does not use harmful types of radiation such as X-rays.

How does it work?

Human tissues contain water, which contain very small particles known as protons that behave like tiny magnets. An MRI machine uses large, powerful magnets to generate a magnetic field that can change how these particles rotate in your body, making them align with the magnetic field. Non-harmful radio waves are then pulsed through the patient, changing the direction of these particles, such that they are no longer aligned with the magnetic field. The radio waves are then turned off, and the particles can then re-align with the magnetic field. Different types of tissue and structures in the body will have particles that re-align differently, which can be detected by the machine to generate a detailed black and white image of the scanned area of the body. In addition to such structural information, MRI scans can provide information about how the brain is wired, levels of important chemicals, blood flow, metabolism, and brain function by acquiring information differently with the same machine.

3D view of an entire human brain taken by MRI, shown from two angles.
3D view of an entire human brain taken by 7 Tesla MRI. Photo courtesy of  B.L. Edlow et al, bioRxiv, 2019.

How do you prepare for an MRI scan?

Since an MRI scan uses a large magnet, electronic devices and metal objects, such as glasses and jewelry, must be removed. There is usually no other preparation required for the scan. Patients must lie very still to generate a clear image. Patients do not need to be sedated, unless they have trouble lying still for the procedure. MRI scans that are obtained for research do not use anaesthesia to avoid unnecessary risk to research participants.

What happens during an MRI scan?

The patient lies down on a table that will move into the tunnel-shaped chamber. The patient is usually awake and will remain in the chamber as several scans are taken during the procedure (about 30-60 minutes). As the scan proceeds, there are often loud mechanical sounds, so earplugs are provided for protection. Some patients may experience claustrophobia, or are bothered by the noises. Becoming more familiar with the procedure, or listening to music or closing your eyes can help alleviate discomfort during the scan.

What do doctors look for in patients with SCAs?

MRI scans are often used to image the brain to detect signs of spinocerebellar ataxia (SCA), especially in a region of the brain known as the cerebellum. SCA is associated with brain cell loss, and appears as reduced volume of brain tissue in the MRI image.

If you would like to learn more about Magnetic Resonance Imaging (MRI), take a look at these resources by the National Institutes of Health and the Mayo Clinic.

Snapshot written by Dr. Claudia Hung and edited by Dr. Gülin Öz.