Craig S. McIntosh

Background and Objectives: Malan syndrome is a rare overgrowth syndrome resulting from NFIX haploinsufficiency due to heterozygous loss-of-function mutations or microdeletions of NFIX on chromosome 19 at p13.2. Phenotypic presentation can vary but is characterized by macrocephaly, long and slender body habitus, skeletal abnormalities, and intellectual disability. Methods: Here, we report on the presentation, management, and development of a patient with Malan syndrome, highlighting the clinical and behavioral aspects of this syndrome, therapeutic interventions employed, and the course of disease over a 15-year period. We review medical records, cytogenetic analysis and neuropsychologic testing results, as well as speech pathology, optometric, and medical reports. In addition, we discuss personalized therapeutic strategies that could potentially be exploited in the future for such overgrowth syndromes. Results: To our knowledge, this is the first longitudinal follow-up report of a case of Malan syndrome to highlight the clinical course, interventions employed, and resulting improvements in neurocognitive function over time. Conclusions: This case highlights the importance of early diagnosis, intervention, and preventative care in overgrowth syndromes, as well as the potential for therapeutic intervention in the future.

Down syndrome is a genetic-based disorder that results from the triplication of chromosome 21, leading to an overexpression of many triplicated genes, including the gene encoding Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 1A (DYRK1A). This protein has been observed to regulate numerous cellular processes, including cell proliferation, cell functioning, differentiation, and apoptosis. Consequently, an overexpression of DYRK1A has been reported to result in cognitive impairment, a key phenotype of individuals with Down syndrome. Therefore, downregulating DYRK1A has been explored as a potential therapeutic strategy for Down syndrome, with promising results observed from in vivo mouse models and human clinical trials that administered epigallocatechin gallate. Current DYRK1A inhibitors target the protein function directly, which tends to exhibit low specificity and selectivity, making them unfeasible for clinical or research purposes. On the other hand, antisense oligonucleotides (ASOs) offer a more selective therapeutic strategy to downregulate DYRK1A expression at the gene transcript level. Advances in ASO research have led to the discovery of numerous chemical modifications that increase ASO potency, specificity, and stability. Recently, several ASOs have been approved by the U.S. Food and Drug Administration to address neuromuscular and neurological conditions, laying the foundation for future ASO therapeutics. The limitations of ASOs, including their high production cost and difficulty delivering to target tissues can be overcome by further advances in ASO design. DYRK1A targeted ASOs could be a viable therapeutic approach to improve the quality of life for individuals with Down syndrome and their families.

Splice modulating antisense oligomers (AOs) are increasingly used to modulate RNA processing. While most are investigated for their use as therapeutics, AOs can also be used for basic research. This study examined their use to investigate internally and terminally truncated proprotein convertase subtilisin/kexin type 9 (PCSK9) protein isoforms. Previous studies have used plasmid or viral-vector-mediated protein overexpression to study different PCSK9 protein isoforms, creating an artificial environment within the cell. Here we designed and tested AOs to remove specific exons that encode for PCSK9 protein domains and produced protein isoforms at more physiologically relevant levels. We evaluated the isoforms’ expression, secretion, and subsequent impact on the low-density lipoprotein (LDL) receptor and its activity in Huh-7 cells. We found that modifying the Cis-His-rich domain by targeting exons 10 or 11 negatively affected LDL receptor activity and hence did not enhance LDL uptake although the levels of LDL receptor were increased. On the other hand, removing the hinge region encoded by exon 8, or a portion of the prodomain encoded by exon 2, have the potential as therapeutics for hypercholesterolemia. Our findings expand the understanding of PCSK9 isoforms and their impact on the LDL receptor and its activity at physiologically relevant concentrations.

Precursor messenger RNA (pre-mRNA) splicing is a fundamental step in eukaryotic gene expression that systematically removes non-coding regions (introns) and ligates coding regions (exons) into a continuous message (mature mRNA). This process is highly regulated and can be highly flexible through a process known as alternative splicing, which allows for several transcripts to arise from a single gene, thereby greatly increasing genetic plasticity and the diversity of proteome. Alternative splicing is particularly prevalent in neuronal cells, where the splicing patterns are continuously changing to maintain cellular homeostasis and promote neurogenesis, migration and synaptic function. The continuous changes in splicing patterns and a high demand on many cis- and trans-splicing factors contribute to the susceptibility of neuronal tissues to splicing defects. The resultant neurodegenerative diseases are a large group of disorders defined by a gradual loss of neurons and a progressive impairment in neuronal function. Several of the most common neurodegenerative diseases involve some form of splicing defect(s), such as Alzheimer’s disease, Parkinson’s disease and spinal muscular atrophy. Our growing understanding of RNA splicing has led to the explosion of research in the field of splice-switching antisense oligonucleotide therapeutics. Here we review our current understanding of the effects alternative splicing has on neuronal differentiation, neuronal migration, synaptic maturation and regulation, as well as the impact on neurodegenerative diseases. We will also review the current landscape of splice-switching antisense oligonucleotides as a therapeutic strategy for a number of common neurodegenerative disorders.

Polyglutamine (polyQ) ataxias are a heterogenous group of neurological disorders all caused by an expanded CAG trinucleotide repeat located in the coding region of each unique causative gene. To date, polyQ ataxias encompass six disorders: spinocerebellar ataxia types 1, 2, 3, 6, 7, and 17 and account for a larger group of disorders simply known as polyglutamine disorders, which also includes Huntington’s disease. These diseases are typically characterised by progressive ataxia, speech and swallowing difficulties, lack of coordination and gait, and are unfortunately fatal in nature, with the exception of SCA6. All the polyQ spinocerebellar ataxias have a hallmark feature of neuronal aggregations and share many common pathogenic mechanisms, such as mitochondrial dysfunction, impaired proteasomal function, and autophagy impairment. Currently, therapeutic options are limited, with no available treatments that slow or halt disease progression. Here, we discuss the common molecular and clinical presentations of polyQ spinocerebellar ataxias. We will also discuss the promising antisense oligonucleotide therapeutics being developed as treatments for these devastating diseases. With recent advancements and therapeutic approvals of various antisense therapies, it is envisioned that some of the studies reviewed may progress into clinical trials and beyond.

Antisense oligomers (AOs) are increasingly being used to modulate RNA splicing in live cells, both for research and for the development of therapeutics. While the most common intended effect of these AOs is to induce skipping of whole exons, rare examples are emerging of AOs that induce skipping of only part of an exon, through activation of an internal cryptic splice site. In this report, we examined seven AO-induced cryptic splice sites in six genes. Five of these cryptic splice sites were discovered through our own experiments, and two originated from other published reports. We modelled the predicted effects of AO binding on the secondary structure of each of the RNA targets, and how these alterations would in turn affect the accessibility of the RNA to splice factors. We observed that a common predicted effect of AO binding was disruption of the exon definition signal within the exon’s excluded segment.

Purpose of review Antisense oligomers (ASOs) have been available for decades: however, only recently have these molecules been applied clinically. This review aims to discuss the possible development of antisense-mediated splice correction therapies as precision medicines for familial hypercholesterolemic patients carrying mutations that compromise normal splicing of the low-density lipoprotein receptor (LDLR) gene transcript. Recent findings Three antisense drugs are currently being assessed in ongoing clinical trials for dyslipidemias, aiming to lower the plasma concentrations of lipoproteins that lead to end-organ damage, principally coronary artery disease. Although a handful of drugs may be applicable to many patients with familial hypercholesterolemia (FH), mutation-specific personalised antisense drugs may be even more effective in selected patients. Currently, there is no therapy that effectively addresses mutations in the LDLR, the major cause of FH. Many mutations in the LDLR that disrupt normal pre-mRNA processing could be applicable to splice correction therapy to restore receptor activity. Summary Precision medicine could provide long-term economic and social benefits if they can be implemented effectively and sustainably. Many mutations found in the LDLR gene could be amendable to therapeutic splice correction and we should consider developing a therapeutic ASO platform for these mutations.

Antisense oligomers (AOs) are increasingly being used for modulating RNA splicing in live cells, both for research and for therapeutic purposes. While the most common intended effect of these AOs is to induce skipping of whole exons, rare examples are emerging of AOs that induce skipping of only part of an exon, through activation of an internal cryptic splice site. In this report, we examined seven such examples of AO-induced cryptic splice site activation – five new examples from our own experiments and three from reports published by others. We modelled the predicted effects that AO binding would have on the secondary structure of each of the RNA targets, and how these alterations would in turn affect the accessibility of the RNA to splice factors. We observed that a common predicted effect of AO binding was a disruption to the exon definition signal within the exon’s excluded segment.

Over 40 diseases, primarily affecting the nervous system, are caused by expansion of simple repetitive sequences found throughout the human genome, termed repeat expansion diseases. Expansions can occur in coding and non-coding regions of the genome, leading to several proposed mechanisms of disease, accumulation of either toxic RNA or toxic protein, although gain-of-function mechanisms are suggested causes of pathogenesis. Currently, there is no cure nor effective treatment strategy for any repeat expansion diseases. However, for many of these expansion diseases, splice-switching antisense oligonucleotides (AOs) may offer promise as a therapeutic strategy, as these compounds have already demonstrated efficacy in the treatment of other types of genetic disorders. Antisense oligonucleotides are short synthetic nucleic acid analogues, designed to target specific pre-mRNA sequences by reverse-complementary Watson-Crick binding, thereby modifying processing and/or abundance of the transcript and the sequence of the encoded protein. While there are a number of applications for AOs, this study focuses on their utility for preventing translation of toxic protein isoforms, either by altering the target transcript to encode a truncated protein isoform, or by disrupting the reading frame to downregulate endogenous protein production. The first part of this study focused on ameliorating the toxic polyglutamine tract found in the ataxin-3 protein that causes spinocerebellar ataxia type 3 (SCA3). One of nine known polyglutamine disorders, SCA3 is a clinically heterogeneous disease, primarily exemplified by progressive ataxia impairing the speech, balance and gait of affected individuals. SCA3 is caused by expansion of a glutamine-encoding tract located at the 5′ end of the penultimate exon (exon 10) of the ATXN3 gene transcript, resulting in conformational changes in ataxin-3 and a toxic gain-of-function. Here, we describe highly efficient removal of the toxic polyglutamine tract of ataxin-3 in vitro by phosphorodiamidate morpholino oligomers (PMOs). Additionally, these PMOs induced a potentially beneficial downregulation of both the expanded iv and non-expanded protein isoforms. As SCA3 has a typical age of onset in the fourth decade, the observed downregulation could delay age of onset by reducing the amounts of the toxic aggregates. Although we induce downregulation of both isoforms, we believe that the proportion of the truncated protein may be sufficient for overall function of ataxin-3, as some studies have shown ataxin-3 protein to be partially dispensable. Recently, several in vitro and in vivo studies have found that targeted knockdown of transcription elongation factors SUPT4H1, and to a lesser extent SUPT5H, can reduce aggregation of expanded transcripts and protein, and alleviate the disease phenotype in animal models of various expansion diseases. We therefore sought to investigate in vitro, the potential of AO-mediated SUPT4H1 downregulation as a therapeutic strategy. We found that our AOs were able to significantly downregulate SUPT4H1, with minimal changes to the rest of the transcriptome. We then assessed whether this downregulation of SUPT4H1 lead to a reduction in expanded ATXN3 mRNA and/or ATXN3 protein expression, however, unfortunately in the models available and under the current study, no modification to the ATXN3 transcript or protein was observed. This lack of effect may be due to the relatively short, expanded repeat lengths in SCA3 cell lines, and we therefore recommend that future studies assess genes with larger expansions, such as the 100-1000s repeat tracts frequently observed in myotonic dystrophy type 1 (DMPK). In order to create an efficient screening process for finding clinic-ready AOs, it is important to have a detailed understanding of the principles of AO design. We therefore present a comprehensive rationale for efficiently design and in vitro delivery of splice modulating AOs. These approaches and recommendations provide a streamlined methodology for any researcher developing AO therapeutics. The results presented in this thesis indicate that morpholino oligomers will provide superior benefit for the treatment of spinocerebellar ataxia type 3, without the toxic effects that result from other antisense oligomer chemistries. Additionally, AO-induced SUPT4H1 knockdown may yet demonstrate therapeutic v application for a multitude of expansion diseases, pending further investigation into the whole transcriptome effects and in vivo efficacy of this strategy. Lastly, our guidelines for therapeutic AO development should aid other researchers in creating the most efficacious and safe AOs for clinical trials. The work presented in this thesis contributes to the greater body of knowledge about the applications of AOs, as well as the need for reliable and systematic protocols in AO research and interpretation. With ongoing collaboration from our industry partners, Sarepta Therapeutics, there is hope that the work presented here will provide a solid foundation for further research into AO therapeutics for the treatment of neurodegenerative expansion diseases.