David Gurzick, PhD
Barb Bailus, PhD

In a recent post, we introduced gene editing technologies with a focus on CRISPR (haven’t seen it? – it’s available on the FAST blog). In this post we will focus on the applications of CRISPR in the gene editing field with specific attention on how this technology can help to cure Angelman syndrome (AS)!

In terms of the number of different research projects sponsored, FAST is the largest single funder of gene modifying therapeutics in Angelman syndrome. While much of this funding is on therapeutic candidates for human translational benefit, a large amount has also been used to support creating a platform to enable university and pharma researchers for high throughput evaluations. Additionally, a large amount of support has been put into helping to determine and develop the best outcome assessments that can be used in clinical trials to help determine if a therapeutic is actually working. This is called a clinical outcome measure assessment (COA).

The testing of these potential therapeutics does not start in humans. Instead, it begins in animals that have a genetic profile consistent with the condition under study. Thankfully, CRISPR technology allows researchers to create animal models (mice, rats, pigs, etc) that have a specific genetic condition, by either removing parts of a gene (deletion), or changing some of the DNA code (mutation). These animals can then be used to test potential therapeutics. FAST has funded the creation of the LEGEND-Rat (Large Deletion UBE3A Rat) using CRISPR technology and an AS pig model. These animal models are especially helpful in evaluating therapeutic benefit, like behavioral (or phenotypic) rescue, to assess for a biomarker that can be used to measure changes after treatment, to assess for effective dosing, and to look at potential treatment toxicity – ensuring that treatments are dosed at appropriate levels. While we often think about the effect of providing too much of something, the opposite is also true; the first doses of penicillin were too low to stop infections. The use of animal models greatly accelerates and reduces the risk when developing promising treatments for the AS community.

Aside from making more animal models, CRISPR can also be used in the lab to edit human cell lines, resulting in accurate and relevant cellular models of various diseases. For AS, this technology allows scientists to study the multiple genetic causes of AS across various cell types with identical genetic backgrounds, culminating in the faster identification of new drug targets and faster evaluation of potential treatments. It is not inconceivable that the near future will permit the use of CRISPR to create personalized models to test multiple types of therapies so as to determine which will work best for a specific patient. The first public project to create a map of the human genome was formally launched in 1990 and after just shy of $3 billion dollars of funding, was declared complete in 2003. At the time of writing this post, 23andMe offers a test for less than $200 that takes longer to arrive via postal mail than it does to sequence. This gives some perspective on how today’s revolutionary advances will be tomorrow’s standard of care treatments.

Beyond use in creating animal models, CRISPR can be used as a gene altering therapy in humans. FAST-funded researchers are working at the forefront of this science, exploring ways to use CRISPR to directly edit the genome inside of a patient, editing a cell’s genome in the lab for transfection into a patient, and to modify the way the genome is read in a patient. While at present these technologies are not quite ready for human clinical trials in Angelman syndrome, they are beginning to appear in human clinical trials for other disorders and have the potential to cause a paradigm shift in how genetic disorders are treated in the very near future. As you read through these gene therapy strategies keep in mind the words of Leah Schust, founder of the The FamilieSCN2A Foundation, who notes, “For gene therapy, each success is all of our success”. These studies all push the advancement of real and meaningful changes to benefit those with AS.

Directly editing a patient’s genome

When most AS caregivers learn about CRISPR for the first time, this is the first avenue that comes to mind. “If there is an error in the UBE3A gene, let’s just go in and fix it.” Well, yes. Though it may not be as simple in practice, the first trials are now underway to treat a common cause of inherited childhood blindness – Leber congenital amaurosis. This marks a breakthrough in the pipeline of therapeutics and one that will be all of our success should it deliver on the anticipated outcomes.

It might be possible to use CRISPR to add in a new “normal” copy of UBE3A, restoring UBE3A expression in the brain, or to inhibit the expression of the paternal antisense transcript and activate the silent copy of the father’s UBE3A gene. With multiple therapeutic avenues, CRISPR could prove to be a highly effective and versatile tool for AS. Dr. Nash and Dr. Jim Wilson, are being funded by FAST to use the human UBE3A gene (hUBE) in mouse models of AS and determine the best construct for a therapeutic. This research involves the direct injection of the hUBE gene into the mouse brain through delivery of adeno-associated virus (AAV). Preliminary work is showing great promise in the recovery of various symptoms and behaviors of AS. This is, in simple terms, the addition of a “new” maternal copy of UBE3A gene into the neurons of the mouse brain, which are missing Ube3a. Think of it like a computer program that has been damaged, maybe you can’t replace the entire computer, but you can add a new version of the program that fixes the problems.

Editing a genome in the lab for relocation into a patient

In Nashville, Tennessee at the Sarah Cannon Research Institute, Victoria Gray received a CRISPR treatment to mitigate, if not resolve, the symptoms of Sickle Cell Disease. In the treatment her blood stem cells were extracted and gene edited in a lab to also produce fetal hemoglobin – a protein no longer created in adults. Following a round of chemotherapy, these modified cells were then reintroduced to her body. This idea bears similarity to the strategy FAST is funding Dr. Joe Anderson at UC Davis to progress. His talk at the 2018 FAST Science Summit provides a detailed description of his approach but in brief it consists of three steps: (1) extracting a sample of blood stem cells from a patient’s body, (2) using a virus (lentivirus) to deliver a new copy of human UBE3A into these blood derived stem cells (hematopoetic stem cells [HSCs]) that will allow these cells to excrete UBE3A, and then (3) insert them intravenously back into the patient for these cells to populate the bone marrow and continuously release cells that have a functional copy of human UBE3A. A curious trait of these stem cells (HSCs) is that they know to home back into the bone marrow, where, in theory, they will create UBE3A expressing blood stem cells for the life of the patient. These blood cells easily cross the blood brain barrier, and they will then be constantly entering the brain, releasing the UBE3A protein that the brain of individuals with AS is missing. This is called “cross-correction”. The neurons of the brain then pick up this UBE3A and use it as if it were its own. Toxicity studies are being prepared and this research is rapidly moving on a course toward submitting an application for an investigational new drug to the FDA. This application process is the first step toward human clinical trials!

Targeting the way the genome is read

In her book, The epigenetics revolution: How modern biology is rewriting our understanding of genetics, disease, and inheritance, Dr. Carey describes the genome like a Shakespeare play (excellent summer reading). The script, or more specifically the words and letters in the play represent DNA. But as anyone who has seen a few Shakespeare plays will note, that script is open for wide interpretation. How the script, the DNA, is read is the realm of epigenetics and those in the AS community know this well. The paternal allele of the 15th chromosome is epigenetically silenced in neuronal cells through methylation. Lots of previous work has targeted this methylation as a means for getting at that precious paternal UBE3A gene to express. Previous studies included diets that sought to promote over-methylation of the UBE3A anti-sense transcript (UBE3A-AS) with the intention that it would decrease UBE3A-AS expression, resulting in increased paternal UBE3A expression. Unfortunately the results did not show a change in methylation status or paternal DNA expression. In a different study using Topetecan, global demethylation was shown and this did result in increased paternal UBE3A expression (sadly with high toxicity and potential off target effects – not so great for long-term use by people, but it has found use in the lab as a tool for comparison testing and proof of concept). Our understanding of controlling and targeting epigenetic changes is rapidly improving and holds the potential to be a viable therapy for AS.

Now, with various gene altering techniques, there are new ways to remove the silencing of paternal UBE3A gene through exploiting this imprinting phenomenon and inhibiting the paternal UBE3A-AS. Two techniques take center stage here, with one presently further along than the other. Starting with the earlier stage approach, we return to CRISPR. Now, most of the time when you hear discussion of CRISPR in the popular press, they are using the term as shorthand for CRISPR/CAS9, the CAS9 being the enzyme that is used to cut DNA. Enter CAS13b. This enzyme does not target the DNA, instead it targets RNA. Following the Shakespeare comparison, it does not target the script, it targets the interpretation. CAS13b allows researchers to point it right at the area that silences paternal UBE3A gene. No one to tell you to be quiet? Talk away. In the lab they refer to this effect as “unsilencing” the UBE3A-AS, which in turns activates the paternal copy of the gene in neurons.

If this sounds complicated, it is. Partly this is because we still do not have consensus among the scientific community around how the silencing of paternal UBE3A gene occurs at the molecular level. The predominant theory is often explained with the analogy of a one lane road upon which is driving a small car (I always picture a VW beetle, but feel free to substitute in your small car of choice). Got that car in your mind? That car is paternal UBE3A moving along and being read off the DNA. Coming in the other direction is a snow plow. It is UBE3A-AS, some RNA that is also being read. If you live in the northern United States you know how this story ends; the plow (UBE3A-AS) pushes the small car off the road and makes it non-drivable. CRISPR/CAS13b looks to resize the UBE3A-AS, so that it no longer collides with the small car. This technology has only come on the research scene within the last several years or so and FAST has collaborated with researchers in both Dr. Segal and Dr. Wilson’s labs to pursue this approach. Other FAST-affiliated researchers, like Dr. Zylka (a member of the FAST Scientific Advisory Board) are also exploring CRISPR techniques to target the UBE3A-AS. An excellent review of his lab’s research in this area, funded by the Angelman Syndrome Foundation is available here. This approach is delivered through viral vector (e.g. AAV), and therefore is permanent and a one-time treatment.

Antisense oligonucleotides (ASOs, or “oglios” as the cool kids call them) perform similar to CRISPR by stopping the UBE3A-AS from colliding into the “small car” of UBE3A gene expression, except it is delivered intermittently through a lumbar puncture into the CSF and, while not a one-time treatment like CRISPR, can be administered to a patient long-term. Nusinersen, or Spinraza, the oft-mentioned miracle drug for Spinal Muscular Atrophy (SMA), is an FDA approved and wildly successful oglio.

Nearly a decade of FAST-funded research in this area, spearheaded by Dr. Dindot and his team at Texas A&M, led to the formation of GeneTx Biotherapeutics. This company was founded by FAST to move this promising technology into to human patients as safely, effectively and quickly as possible. This was done with world renowned experts in ASO drug development. In February of 2018, it was announced that GeneTx is conducting all the necessary studies, protocol development, and professional requirements to secure investigational new drug status with the U.S. Food and Drug Administration, and in March of 2018 GeneTx received Orphan Drug Designation for GTX-102, an ASO for the treatment of Angelman syndrome. This the penultimate step before human clinical trials (the clinical trial pathway was the topic of a previous post. In August of 2019, an exclusive option agreement was made between GeneTx and Ultragenyx Pharmaceutical, Inc. to advance GTX-102, the investigational ASO created by GeneTx and in September of 2019 this drug was awarded with Rare Pediatric Disease Designation. These awards acknowledge the pressing need for therapeutics for AS. Roche and other biopharma companies, who are working with FAST through the Angelman Biomarkers and Outcome Measures Alliance, have also revealed their research toward ASOs for AS.

A bold path forward

All of the promising gene therapy options, including those that are CRISPR-based, have a few important challenges to overcome. These include how the CRISPR is delivered, the way it is distributed throughout the neurons of the brain, the potential of off-target effects, and the potential immune response. Some of these challenges are being addressed by a new initiative from the U.S. National Institutes of Health (NIH) to improve gene editing approaches towards reducing the burden of diseases caused by genetic change. This new initiative has multiple prongs, with several laboratories focusing on designing new CRISPR proteins, new delivery methods, detecting adverse effects and evaluating CRISPR in various animal models including rodents, pigs and non-human primates. FAST-fire team member Dr. Segal at UC Davis is part of a multimillion dollar grant to evaluate these CRISPR proteins in non-human primates. Dr. Segal will be a co-director of this program at UCD, and details on this story will be featured in an upcoming blog. This development is extremely exciting for the genetic disease field, as CRISPR offers one of the best possible avenues for a cure. Having the NIH spearhead such an extensive program means that a large portion of the work to bring these potential gene editing proteins to the clinic will be government funded, decreasing the burden on individual disease foundations while also bringing the best scientists together to accelerate a path forward.

As of today, there are multiple CRISPR related clinical trials in various stages of progress listed on the USA government clinical trials site (check out the growing list); it is not hard to imagine that this will exponentially increase in the next several years. Beyond government clinical trials, multiple companies have formed or hold patents on CRISPR technology, including several publicly traded companies: CRISPR Therapeutics, Editas, Caribou, and Recombinetics. As these companies navigate human clinical trials, they will lay a path for others to follow in the near future. The use of CRISPR to treat AS is currently in the pre-clinical stages, but progress – rapid progress – is being made to advance CRISPR-based therapies for AS toward Phase I human clinical trials. We are all in for an exciting ride, one that could change the medical field as dramatically as the discovery of penicillin over 90 years ago, and FAST is making sure we have the best team to arrive at a pinnacle successfully!