Gene Therapy for Dravet Syndrome: State-of-the-Field
Several years ago, a genetic therapy still seemed a distant possibility for Dravet syndrome. However, advances to our understanding of genetics and medicine have allowed researchers to develop new approaches to gene-based interventions, bringing the possibility of disease-modifying therapies closer to reality for Dravet syndrome.
In 80-90% of cases or more, Dravet syndrome is caused by a mutation in one copy of SCN1A, a gene that encodes a specific sodium channel, called Nav1.1, which is particularly important for some cells in the brain to communicate.1–3 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.4 One straightforward approach to a genetic therapy for a haploinsufficiency would be to deliver a replacement copy of the healthy gene. However, a major hurdle in the case of SCN1A is that the gene is very large and cannot fit inside the delivery vehicles (called vectors) that are currently utilized in genetic therapies. In addition to the difficulty of the gene size, a genetic therapy for Dravet syndrome would need to be delivered to cells in the brain, which can be more challenging to reach than some of the other organs in the body. Despite these challenges, researchers have discovered several creative solutions to overcome the haploinsufficiency that causes Dravet syndrome.
Understanding DNA-RNA-PROTEIN - A Recipe Based Analogy
Sometimes in the past when I have been trying to explain the idea of DNA, RNA, and proteins to friends and family who are not well-versed in molecular biology, I used the example of a recipe blog. I think many of us have come across recipe blogs posts, that are full of extra 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 within DNA leads to a protein that has a role in our body’s function.
I think of the entire recipe 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 this analogy is often buried within the recipe blog post. Cells make a “transcript” from the DNA (in a process called transcription) that just contains the most important information for making the eventual gene product- called a protein. Cells follow instructions laid out in the DNA about where the gene “recipe” begins and ends and splice out information that is not 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. Similar to the recipe analogy, the main blog (DNA/gene) is always housed on a website, but you can handwrite or 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, and would be equivalent to actually cooking the recipe. In the case of SCN1A, the mRNA contains the recipe to make a sodium channel called Nav1.1. 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 Veronica Hood, PhD; DSF Scientific Director
Targeting DNA Regulation with Genetic Therapies
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 above.> 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 tune 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 affect the regulatory regions to increase expression of SCN1A.
ETX101. Encoded Therapeutics has developed an approach to increase expression of SCN1A which they have called ETX101. Instead of delivering a new copy of SCN1A, ETX101 delivers a regulatory gene that acts to increase expression of SCN1A. The gene that ETX101 will be delivering to cells is called an engineered transcription factor. Since this engineered transcription factor is much smaller than the SCN1A gene, ETX101 can fit within a commonly used delivery vector, called 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 neurons, called inhibitory interneurons, reducing or eliminating seizures and other symptoms and comorbidities of Dravet syndrome. The preclinical work for ETX101 was published in 2022 and provided evidence that this approach could increase SCN1A expression, decrease seizures, and increase survival in a mouse model of Dravet syndrome. They additionally showed in non-human primates that ETX101 was well-tolerated and delivered broadly across the brain.5 Encoded Therapeutics is working towards a clinical trial for ETX101, called the ENDEAVOR Study, with hopes to begin by 2023.
CRISPR. CRISPR technology has gained a lot of notoriety in recent years. 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 mutations in DNA. Notably, this would still be a challenging approach for Dravet syndrome with the current technology given the size of the SCN1A gene and broad spread of mutations across the entire gene within the patient population. However, some researchers have been utilizing CRISPR technology in a different way to increase gene expression. Traditionally, ‘CRISPR associated protein 9’ or “Cas9”, is used in conjunction with a “guide RNA sequence” to locate the target DNA sequence and make a cut. Scientists have created 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. This approach is also referred to as ‘CRISPR activation’ or ‘CRISPRa.’ 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 SCN1A.6,7 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 challenges to the delivery method and efficiency of increasing gene expression. One of these studies used injection of multiple delivery vectors to contain both the dCas9 and the guide RNA sequences with limited expression in the brain, and the other study took 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 with Genetic Therapies
Regulation of expression can also occur at the RNA-level. Scientists are taking advantage of some of those regulatory instructions to increase the amount of Nav1.1 that 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 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 mRNA 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 Nav1.1.8 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 viral vectors might. The other advantage to this approach is that only the cells that should be naturally expressing SCN1A 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 mouse models of Dravet syndrome.9,10 Clinical trials (called the MONARCH Study in the US) began in late 2020 for STK-001 to determine the safety, pharmacokinetics, and efficacy in patients with Dravet syndrome. STK-001 is not a permanent or one-time therapy; instead, STK-001 will likely need to be administered every several months and the current trial will aim to identify the optimal dosing schedule. As of reports from July 2022, the trials have been progressing safely with some early indications of seizure reduction.
tRNA. Tevard Biosciences (who partnered with Zogenix, now a part of UCB in late 2020) is working to advance two therapies that could correct the SCN1A haploinsufficiency in Dravet syndrome. They use a different kind of RNA, called transfer RNA or tRNA, to increase SCN1A expression. The first approach specifically targets 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 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 nonsense mutation by reading through the premature stop signal and allowing the Nav1.1 protein to be made correctly.11 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, making them a one-time therapy. In addition to these tRNA approaches, in the spring of 2022, Tevard also added a third approach to their developmental pipeline, called an mRNA-amplifier, to increase expression from mRNA strands.12 Currently, the preclinical experiments have all been done 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 a lot of proof-of-concept work that will have to be done.
CMP-SCN. The company CAMP4 is working to develop their pipeline therapeutic, CAMP-SCN that works by targeting a naturally occurring regulation for SCN1A. Normally, there are some special RNA strands that bind to SCN1A mRNA to “turn-down” the expression. The approach of CMP-SCN is to decrease the regulatory RNA so that the SCN1A mRNA from the healthy copy of the gene can be free to make additional Nav1.1 sodium channels. In mice, a similar approach (modified to match mouse genetics) was effective at reducing seizures and normalizing some electrical activity in cells of the brain.13
Focusing on Alternative Approaches to Gene Replacement
As mentioned above, one of the major challenges to overcome for gene therapy in Dravet syndrome has been the size of SCN1A, which is too large to 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.14,15 All of these studies are still in early stages, but the work in cells and mouse models have 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.
Targeting Genes that Can Compensate for SCN1A Haploinsufficiency
A few researchers have attempted to compensate for the decreased expression of SCN1A by altering other genes in the same pathway. One group sent in another copy of a gene (NaV1B) that interacts with Nav1.1, with mixed results based on sex in a mouse model of Dravet syndrome and modest impacts on seizures.16 Another group has decreased expression of a separate sodium channel gene, SCN8A, which eliminated behavioral (motor) seizures (for 5 months post-treatment) and reduced mortality in a mouse model of Dravet syndrome.17 In 2021, DSF awarded a post-doctoral fellowship to a researcher working on this approach.
In summary, there are several disease-modifying therapeutic approaches in various stages of development for Dravet syndrome. It is encouraging to see so many different tactics being employed to overcome the challenges to correcting the haploinsufficiency of SCN1A in Dravet syndrome, and the future of these potentially disease-modifying therapies is bright.
Learn more about the genetics of Dravet syndrome.
More questions about this topic? Email DSF’s Scientific Director, Veronica Hood, PhD: email@example.com
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