Gene-based therapies: overview and application to chromosome 15q

Precision therapeutics. Ongoing research in precision therapies in neurological disorders, including 15q-related disorders, is occurring in three spaces: 1) gene therapy, 2) anti-sense oligonucleotides (ASOs), and 3) small molecules (repurposing existing drugs or generating new drugs), where the latter is primarily focused on addressing the symptoms of genetic disorders (i.e. seizures) rather than the cause (i.e gene dysfunction). Each of these forms of therapy has particular challenges, including, critically, the delivery method. The blood-brain barrier (doing its job well) restricts the access of large or hydrophilic medications to the central nervous system (CNS), therefore scientists building these drugs must not only consider efficacy and safety of the drug itself, but also efficacy and safety of the delivery method to the CNS. Below we explore ASOs and gene therapies and their application in 15q-related disorders in more depth. We will not discuss small molecule therapies here as the topic is too broad in scope for the purposes of this post, and we would like to focus primarily on genetically-based therapies.

Figure 1. This figure from Amado and Davidson (2021) shows examples of the targeted therapies described in this post. This includes antisense oligonucleotides (A) and viral-based silencing of a gain of function allele (B) or supplementation of a loss of function allele (C). The figure is focused on therapies for amyotrophic lateral sclerosis, while this blog is focused on syndromes resulting from CNVs in 15q11-13; however, the overall principles are similar.

Gene therapy. Gene therapy either 1) introduces new genetic material (“gene supplementation”), where the healthy version of a gene is housed in a viral vector then injected into the central nervous system (CNS), or 2) modulates existing genetic material via CRISPR/Cas9-like techniques.

  • Supplemental gene therapies are ideally suited for genetic disorders resulting from haploinsufficiency, often due to a protein-truncating variant. Examples of such disorders include Dravet Syndrome due to a loss-of-function variant in SCN1A. There are at least 3 preclinical trials for UBE3A replacement trials ongoing for Angelman Syndrome.
  • The CRISPR/Cas9 system is a gene editing tool that essentially acts as a homing device attached to a scissor, along with new data ready to be inputted, all targeting a specific genetic sequence. CRISPR/Cas9 methods may be employed to modulate the expression of genes by targeting their promoter regions, often to inactivate genes. In imprinting disorders such as Angelman Syndrome, CRISPR/Cas9 can be employed to demethylate the paternally-inherited UBE3A, thereby un-silencing the gene – or allowing the gene to be transcribed.

Gene therapies have the potential for a long-lasting effect with a single treatment. The question of delivery method is a significant hurdle in CNS gene therapy. The capacity of the vector versus the size of the gene poses a considerable challenge (for example, regarding gene supplementation – the SCN1A gene is larger than the capacity of the AAV vector to contain it); furthermore, the viral vectors themselves may be toxic to the CNS, or may cause inflammation due to an immune reaction. Potential disadvantages of such therapies include potential for severe immunogenicity, off-target irreversible effects, and unwanted mutations. Finally, dosing is not clear for the administration of such medications – where it is possible to monitor levels for many anti-seizure medications, there are no clear biomarkers for monitoring either gene therapy efficacy or pre-toxicity at this point – furthermore, since only one dose is administered, there is no room for error.  Further complicating matters, dosing may vary for different disorders, may differ according to administration/delivery method, and dosing requirements may vary in different individuals. The line between toxic and therapeutic is far from clear.

Antisense oligonucleotides (ASOs). ASOs are small single-stranded nucleic acid sequences that target complementary strands of mRNA to alter gene expression via modulation of pre-mRNA splicing or regulation of translation. These therapies may be particularly useful in genetic disorders caused by gain-of-function variants, such as those seen in SCN2Arelated early infantile epileptic encephalopathy or KCNT1-associated epilepsy of infancy with migrating focal seizures, for example. As such, there is a theoretical role for ASO therapy in treating 15q duplication syndrome – though there may be a risk in excessive downregulation of implicated genes such as UBE3A, since Dup15q’s counterpart, Angelman Syndrome, is due to the lack of the functional UBE3A gene transcript. While ASOs are typically utilized to downregulate gene expression, they may also be instrumentalized to upregulate gene expression as well, via Targeted augmentation of nuclear gene output (TANGO) (this is one strategy for Dravet Syndrome, for example). Therefore ASOs may also play a role in treating disorders due to loss-of-function variants. Additionally, and similarly to the use of gene therapies in imprinting disorders, ASOs may be used to “unsilence” genes – there are, in fact, at least three trials ongoing to explore the use of ASOs to activate the paternal UBE3A gene. Importantly, ASOs do not introduce new genetic material or alter the genetic code itself in any way; thus patients require repeated treatments to maintain therapeutic effect. Furthermore, in order to overcome the blood-brain barrier, delivery is by intrathecal injection. There is potential for immunogenicity, and use of ASOs in neurological disorders may be limited in part by the challenges posed by the blood-brain barrier. Similar to gene therapy, dosing requirements are largely unknown and may vary depending on the strategy of administration and the particular therapies in question.

Existing precision therapies for neurological conditions. Currently, we have approved ASOs and gene therapies for two neuromuscular disorders that are invariably fatal at a young age, Spinal muscular atrophy (SMA) and Duchenne muscular dystrophy. There are a handful of available ASOs that drive exon skipping in the dystrophin gene, thereby resulting in a functionally intact, if smaller, dystrophin protein; these may only be utilized with specific variants, however. There is also ongoing work to create a microdystrophin gene that could fit into an AAV vector for systemic administration – though there have been serious safety concerns with trials thus far, which has slowed their progress. There are three approved genetic-based therapies for SMA, including Nusinersen (Spinraza), an ASO that stabilizes SMN2, the sister gene for SMN1, and Onasemnogene Abeparvovec (Zolgensma), a gene therapy that introduces an intact SMN1 transgene, thereby restoring the SMN1 protein. There are also many other trials ongoing for other neurological disorders. Here we also note that there are researchers working on personalized ASO or gene therapies for individuals with rare and often devastating neurological conditions. These conditions are so rare that clinical trials are impractical or unfeasible. There is, therefore, an even greater responsibility for researchers to ensure safety and tolerability prior to pursuing experimental therapies that hold a promise of a cure. There must be clear protocols in place for dosing, monitoring, and management in the case of adverse effects. Recent n of 1 trials in this space have resulted in severe, irreversible neurological consequences including hydrocephalus and death.

Reconsidering our goals. Perhaps we should more deeply consider the goals of drug development for rare genetic neurological conditions. Many clinicians and families will state that of course, the goal is a cure, and along with a cure, we seek advancement in medicine as a whole; we seek to broaden the scope of science to treat and perhaps even eradicate devastating neurological conditions. But I urge us to be more nuanced as we consider the consequences of these therapies, the potential for false hope, and the potential for irreversible loss. Based on animal studies, it appears that many experimental therapies will result in the reduction of seizures, but we do not yet know how this effect will persist at different ages, when gene expression across age groups may vary (this is particularly relevant, for instance, in SCN1A-related disorders).  Furthermore, it is not clear to what extent gene therapies may reverse or alter already established neurodevelopmental phenotypes including intellectual disability, ADHD-like features, or autistic features; stated differently, it is not clear how these therapies may influence future learning, cognition, language development, and communication – or how they might alter personality, identity, and personhood. Cases like the infamous death of Jesse Gelsinger also remind us that a lack of careful controls can not only lead to adverse outcomes and even death, but a complete stagnation in the field of gene therapy as a whole.

What to know. The creativity of scientists and clinicians researching gene-based therapies, the rapidity by which therapies are being explored, and their promise for treating severe genetic epilepsies is simply staggering. In the field of 15q-related disorders, and Angelman Syndrome in particular, there are several promising therapies advancing through preclinical and clinical trials. However, we urge caution when it comes to experimental gene-based therapies, especially where protocolized clinical trials may not be feasible at this time. We do not yet know the range of potentially harmful and/or irreversible side effects that individuals may experience with ASO or gene therapies, and clinical trials are limited by small patient numbers. Appropriate dosing of these medications is unclear, and there is risk for immunogenicity with introduction of these therapies, which may limit the ability of patients to participate in future promising trials. Importantly, we must ask, who are we treating, and what are our goals? Are we delivering treatments equitably across different socioeconomic groups, races, and ethnicities? What are the potential benefits and the risks? It is as if we are squinting through a dense forest toward a singular promise: a cure, the restoration of neurons to a state of healthy functioning – but at times in our eagerness, we are willfully trodding through the undergrowth, unsure if we are even on the right path. We must be bold and continue to have great hope in order to progress – but I urge the community of scientists, families, and clinicians to keep both eyes open, to tread carefully, and to look both forward and backward, keeping in mind the gravity of past errors and the potential for unintended consequences. In time, we will see astronomical growth. Let us ensure that this advancement is just, equitable, and both compassionate and respectful to all of our patients and families.

Alexis Karlin

Alexis Karlin is a pediatric neurologist at the Children’s Hospital of Philadelphia.