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Several years ago, gene therapy still seemed a distant possibility for Dravet syndrome. While preclinical and clinical gene therapy approaches were marching forward for other diseases, the size of the SCN1A gene hampered progress on traditional approaches to gene therapy for Dravet syndrome. However, advances to our basic understanding of genetics, along with an ever-expanding
“genetic toolset,” have allowed researchers to develop new approaches to gene-based interventions, making truly disease-modifying therapies closer to a reality for Dravet syndrome.

In more than 80% of cases, Dravet syndrome is caused by a mutation in one copy of the SCN1A gene that encodes a sodium channel, Nav1.1 (Zuberi et al 2011, Wu et al 2015). Mutations in SCN1A that are associated with Dravet syndrome result in about 50% decreased expression or function of the Nav1.1 sodium channel (see Figure 1; ). This type of reduction in gene expression is referred to as a haploinsufficiency (Catteral et al 2010). Traditional gene therapy approaches to haploinsufficiency would be gene replacement. Gene replacement therapy uses the casing of a virus, a viral vector, to deliver DNA that encodes a healthy copy of the mutated gene to cells. However, this approach has proven difficult in Dravet syndrome because the SCN1A gene is quite large and that amount of DNA cannot fit in commonly used delivery vectors. Researchers are now circumventing this problem by delivering other genes that can increase the expression of the unaffected copy of SCN1A, targeting the gene at the RNA-level instead of the DNA, or optimizing other kinds of larger delivery vectors. In addition to the problem of the gene size, delivery to specific subsets of neurons in the brain is another challenge that researchers are working to overcome.

DNA-RNA-PROTEIN - A Recipe Based Analogy

Sometimes in the past when I’ve been trying to explain the progression of DNA-RNA-protein to friends and family who are not well-versed in molecular biology, I’ve used the example of a recipe blog. I think many of us have come across recipe blogs posts, that are full of extraneous tidbits about the background of a recipe with the actual recipe being almost hidden within the blog. Sometimes these blogs are infuriating when you just want to find a recipe, but today we will try to make use of recipe blogs as an analogy to understand each step of how a gene makes a protein.

I think of the entire blog as our DNA. The blog then has individual blog posts detailing a recipe (the genes). Just like a recipe blog post has a lot of extra details that tell you all about the background of the recipe, there is a lot of other information in the DNA surrounding a gene that informs which cells should use the gene, when they should use the gene, how they might modify the gene for different situations, and how much of the gene to express. Each gene would be an individual recipe blog post about making one specific recipe.

Now the RNA, is really the actual “recipe,” which in our analogy is often buried within the recipe blog post. Cells make a “transcript” from the DNA (a process called transcription) that just contains the most important information for making the eventual gene product- a protein. They follow instructions laid out in the DNA about where the gene “recipe” begins and ends, and they splice out information that isn’t necessary to the recipe. Some genes use alternative splicing which allows a few variations on the recipe to be made from one gene; an analogy might be a recipe for bread where you swap add ins like garlic or nuts to get a slightly varied ending product. The RNA transcript “recipe”, called messenger RNA (MRNA) then can be used by the cell as instructions to make the protein product. The DNA always stays protected in the nucleus of the cell, but the mRNA goes out into the cell to be read, usually multiple times. So back to the recipe blog analogy: the main blog (DNA/gene) is always housed on that website, but you can print off the core recipe (mRNA) and use it yourself multiple times to actually make the recipe.

Finally, the RNA “recipe” is used to make a protein. This biological process is called translation. You can think of this as actually cooking the recipe. In the case of SCN1A, the mRNA contains the recipe to make a sodium channel, Navl.1. This sodium channel is used primarily by inhibitory neurons in the brain that help to calm electrical activity used in neuronal communication (See Figure 1 for more details).

Written by Dr. Veronica Hood, DSF Scientific Director

Targeting DNA Regulation

When a gene within your DNA (like SCN1A) is expressed, the first step is to make a strand of messenger RNA (mRNA) that can then be used as instructions to make the final product: a protein (the sodium channel, Nav1.1). <If you need a quick overview on DNA-RNA-protein, check out the analogy in Box 1.> In order for a specific cell to know when and how much of a gene to express, there are regulatory regions in the DNA (often called promoters or enhancers) that act as markers to control gene expression. Molecules that bind to these regulatory regions and turn gene expression on, off, up, or down, are called “transcription factors,” (because the process of making mRNA from DNA is called “transcription”). Researchers have been working to identify where these regulatory regions are for SCN1A and ways that they can modulate the activity of the regulatory regions to increase expression of SCN1A.

ETX101Encoded Therapeutics has developed an approach to increase expression of the SCN1A gene with a new gene therapy called ETX101. Instead of delivering a new copy of SCN1A, ETX101 delivers a regulatory gene that acts to increase expression of SCN1A and, in turn, the sodium channel Nav1.1. The gene that ETX101 will be delivering to cells is an engineered transcription factor that will help to increase the expression of the SCN1A gene. Because this engineered transcription factor is much smaller than the SCN1A gene, ETX101 can be packaged within an adeno-associated viral (AAV) vector for delivery to cells. ETX101 is anticipated to be a one-time-treatment delivered directly to the brain, where the engineered transcription factor will be expressed in the major type of neurons that utilize the SCN1A gene. The hope is that this will restore the function of these inhibitory interneurons, ameliorating seizures and other comorbidities, and preclinical work presented at scientific meetings has shown this approach to be effective in rodent models of Dravet syndrome. Additionally, injection of ETX101 to the brain of non-human primates shows broad distribution of ETX101 and favorable safety outcomes. Encoded Therapeutics hopes to begin a clinical trial, called ENDEAVOR, for ETX101 in patients with Dravet syndrome later in 2021.

CRISPR. Another approach currently being tested in preclinical cell and rodent models is also targeting the regulation of the SCN1A gene. Commonly, CRISPR technology is used to “cut and paste” sequences of DNA, and the therapeutic potential has been largely focused on the ability to cut out and correct a specific mutation in a gene. However, some researchers have been utilizing this technology in a different way to increase gene expression. ‘CRISPR associated protein 9,’ or Cas9, is used in conjunction with a “guide RNA sequence” to locate the target DNA segment and make a cut. A deactivated version of Cas9, called dCas9, no longer harbors the ability to cut DNA, but instead can be connected to molecules that increase gene expression. Several groups of academic researchers are investigating how this technology could be utilized to increase SCN1A expression by targeting dCas9 to specific regulatory regions for the SCN1A gene (Colasante et al 2020, Yamagata et al 2020). Work in cell lines and mouse models of Dravet syndrome have shown the effectiveness of this approach to increase SCN1A, and consequently levels of the Nav1.1 sodium channel. Additionally, experiments indicate that this treatment approach can improve neuronal communication and seizure activity in Dravet syndrome mice. While encouraging, this work is still in preclinical development; there are still challenges to the delivery method and efficiency of increasing gene expression. The current experiments used injection of multiple delivery vectors to contain both the dCas9 and the guide RNA sequences with limited expression in the brain, or they have taken advantage of mouse genetics to ensure the efficient delivery to the correct cells. Despite the need for advancements to the technology for eventual human therapies, these proof-of-concept studies highlight that this approach could correct the haploinsufficiency of SCN1A and improve patient outcomes.

Targeting RNA Regulation

A lot of regulation can occur at the RNA level as well. Scientists are taking advantage of some of those regulatory instructions to increase the amount of Nav1.1 that the SCN1A mRNA creates by targeting alternative splicing, correction of nonsense mutations, and stabilization of SCN1A mRNA transcripts.

TANGO ASO. Stoke Therapeutics has developed a strategy for a disease-modifying approach called STK-001 that works at the level of RNA-splicing. RNA-splicing occurs to remove the sections copied from the DNA code that are not essential to the “recipe” for creating the protein product (in this case, Nav1.1). The SCN1A gene sometimes includes a section of the DNA code, called a poison exon, within the RNA transcript that tells the cell to trash the strand of RNA instead of using it to produce the Nav1.1 protein. Stoke Therapeutics approach, called Target Augmentation of Nuclear Gene Output (TANGO), sends in a small piece of RNA that blocks the inclusion of the poison exon, and thus, increases the amount of RNA strands that produce Nav1.1 (Lim et al 2020). The small piece of RNA, called an antisense oligonucleotide or ASO, can be packaged inside a lipid droplet that allows the ASO access into cells. This type of packaging is ideal, as it does not pose the same risks of off-target immune reactions as some other delivery methods. The other advantage to this approach is that only the cells that should be naturally expressing the SCN1A gene will be affected by the therapy, helping to reduce off-target effects. STK-001 is delivered by intrathecal injection (similar to a lumbar puncture or an epidural). Preclinical work showed efficacy to reduce seizures and mortality in a mouse model of Dravet syndrome (Han et al 2020). Clinical trials (called MONARCH and SWALLOWTAIL) began in late 2020 and early 2021 for STK-001 to determine the safety, pharmacokinetics, and efficacy in patients with Dravet syndrome. The trials will determine how often STK-001 needs to be administered; it is thought potentially STK-001 administration will be needed every several months, as ASO’s are eventually broken down by the cells that take them up. We expect to hear the first reports on the STK-001 trials by the end of 2021.

tRNA. Tevard Biosciences recently partnered with Zogenix, now a part of UCB, to advance two therapies that could correct the haploinsufficiency in Dravet syndrome. They are using a different kind of RNA, called transfer RNA or tRNA, to increase SCN1A gene expression. They are developing two different approaches. The first therapy will specifically target nonsense mutations. Nonsense mutations create a change in the DNA code that tells the cell to prematurely stop making the Nav1.1 protein, leading to a shortened version that either gets broken down by the cell or does not work as efficiently as it should. The therapy in development uses a tRNA that can overcome the mutation and allow the Nav1.1 protein to be made correctly. The other therapy they are developing also uses tRNA, but this approach helps to stabilize the SCN1A mRNA so that it can be used to create more copies of the Nav1.1 protein. Both of these approaches would be delivered in an AAV vector to cells in the brain. Currently, these experiments have all been shown in cells, and the company is now working with animal models of Dravet syndrome. These therapies are exciting because of the potential they hold for broad application to other genes, but there is still work to be done in animal models.

Focusing on Gene Replacement

As mentioned above, one of the major challenges to overcome for gene therapy in Dravet syndrome has been the large size of the SCN1A gene that does not fit into the vectors that would be most ideal for delivery to neurons in the brain. Several academic research collaborations are working on utilizing larger types of adenoviral vectors to deliver a replacement copy of the SCN1A gene. There have also been some groups working on splitting a replacement gene into two vectors that deliver the gene to the cell where it can reassemble to encode for the full Nav1.1 protein. All of these studies are still in early stages, but some work in cells and mouse models is beginning to show promising results. It is yet to be determined what the challenges of using these gene replacement approaches in humans might be, but the field is marching forward steadily.

In summary, there are several disease-modifying therapeutic approaches in various stages of development. It is encouraging to see so many different tactics being employed to overcome the challenges to correcting the haploinsufficiency of SCN1A in Dravet syndrome. With so many talented minds pushing forward from so many different angles, there is a lot of hope for the development of a therapy that can truly treat the root cause of Dravet syndrome and dramatically improve the outlook for patients.

Learn more about the genetics of Dravet syndrome.

More questions about this topic? Email DSF’s Scientific Director, Veronica Hood: veronica@dravetfoundation.org

Catteral et al (2010) Journal of Physiology. DOI: 10.1113/jphysiol.2010.187484
Colasante et al (2020) Molecular Therapy. 
DOI: 10.1016/j.ymthe.2019.08.018
Han et al (2020) Science Translational Medicine. 
DOI: 10.1126/scitranslmed.aaz6100
Lim et al (2020) Nature Communications. DOI: 10.1038/s41467-020-17093-9
Wu et al (2015) PEDIATRICS. DOI: 10.1542/peds.2015-1807
Yamagata et al (2020) Neurobiology of Disease. 
DOI: 10.1016/j.nbd.2020.104954
Zuberi et al (2011) Neurology. DOI: 10.1212/WNL.0b013e31820c309b

Updated March 2021

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