Dr May Aung-Htut

Overgrowth-intellectual disability (OGID) syndromes encompass a group of rare neurodevelopmental disorders that frequently share common clinical presentations. Although the genetic causes of many OGID syndromes are now known, we lack a clear mechanistic understanding of how such variants disrupt developmental processes and ultimately culminate in overgrowth and neurological symptoms. Patient advocacy groups, such as the Overgrowth Syndromes Alliance (OSA), are mobilising patients, families, clinicians and researchers to work together towards a deeper understanding of the clinical needs of patients with OGID, as well as to understand the fundamental biology of the relevant genes, with the goal of developing treatments. In this Review, we summarise three OGID syndromes encompassed by the OSA, namely Sotos syndrome, Malan syndrome and Tatton-Brown-Rahman syndrome. We discuss similarities and differences in the biology behind each disorder and explore future approaches that could potentially provide a way to ameliorate some of the unmet clinical needs of patients with OGID.

Background
Apolipoprotein C-III (APOC3) plays a crucial role in triglyceride metabolism and is closely associated with cardiovascular disease risk. Elevated APOC3 levels contribute to higher plasma triglycerides and increased risk of atherosclerosis, making APOC3 expression an attractive and logical therapeutic target.

Methods
While studying various APOC3 transcript isoforms expressed in hepatoma cell lines (HepG2, Huh7) and healthy liver tissue using publicly available long-read RNA sequencing, we found three novel APOC3 isoforms. These isoforms were validated through RT-PCR and Sanger sequencing.

Results
All three novel isoforms are splicing variants of the MANE transcript, APOC3-201. Isoforms 1 and 2 exhibit splicing patterns similar to APOC3-201 from exons 2–4; however, isoform 1 shares its exon 1 splicing pattern with APOC3-203, while isoform 2 features an extended exon 1 that includes exon 1a, the adjacent intronic region, and exon 1b. The third isoform closely resembles APOC3-201, but lacks exon 2, which contains the translation start codon. Remarkably, similar APOC3 splicing patterns and transcript variants were observed in Caco-2 cells, a model of the small intestine, indicating that these isoforms are not liver-specific.

Conclusions
This study identifies three novel APOC3 isoforms and highlights their expression in both hepatic and intestinal cell models. Further studies are needed to elucidate the functional roles of these novel isoforms and their contribution to the regulation of APOC3 gene expression.

Vascular Ehlers–Danlos syndrome or Ehlers–Danlos syndrome type IV (vEDS) is a connective tissue disorder characterised by skin hyperextensibility, joint hypermobility and fatal vascular rupture caused by COL3A1 mutations that affect collagen III expression, homo-trimer assembly and secretion. Along with collagens I, II, V and XI, collagen III plays an important role in the extracellular matrix, particularly in the inner organs. To date, only symptomatic treatment for vEDS patients is available. Fibroblasts derived from vEDS patients carrying dominant negative and/or haploinsufficiency mutations in COL3A1 deposit reduced collagen III in the extracellular matrix. This study explored the potential of an antisense oligonucleotide (ASO)-mediated splice modulating strategy to bypass disease-causing COL3A1 mutations reported in the in-frame exons 10 and 15. Antisense oligonucleotides designed to redirect COL3A1 pre-mRNA processing and excise exons 10 or 15 were transfected into dermal fibroblasts derived from vEDS patients and a healthy control subject. Efficient exon 10 or 15 excision from the mature COL3A1 mRNA was achieved and intracellular collagen III expression was increased after treatment with ASOs; however, collagen III deposition into the extracellular matrix was reduced in patient cells. The region encoded by exon 10 includes a glycosylation site, and exon 15 encodes hydroxyproline and hydroxylysine-containing triplet repeats, predicted to be crucial for collagen III assembly. These results emphasize the importance of post-translational modification for collagen III homo-trimer assembly. In conclusion, while efficient skipping of target COL3A1 exons was achieved, the induced collagen III isoforms generated showed defects in extracellular matrix formation. While therapeutic ASO-mediated exon skipping is not indicated for the patients in this study, the observations are restricted to exons 10 and 15 and may not be applicable to other collagen III in-frame exons.

Rare diseases affect almost 500 million people globally, predominantly impacting children and often leading to significantly impaired quality of life and high treatment costs. While significant contributions have been made to develop effective treatments for those with rare diseases, more rapid drug discovery strategies are needed. Therapeutic antisense oligonucleotides can modulate target gene expression with high specificity through various mechanisms determined by base sequences and chemical modifications; and have shown efficacy in clinical trials for a few rare neurological conditions. Therefore, this review will focus on the applications of antisense oligonucleotides, in particular splice-switching antisense oligomers as promising therapeutics for rare neurological diseases, with key examples of Duchenne muscular dystrophy and spinal muscular atrophy. Challenges and future perspectives in developing antisense therapeutics for rare conditions including target discovery, antisense chemical modifications, animal models for therapeutic validations, and clinical trial designs will also be briefly discussed.

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.

Dysferlin is a large transmembrane protein involved in critical cellular processes including membrane repair and vesicle fusion. Mutations in the dysferlin gene (DYSF) can result in rare forms of muscular dystrophy; Miyoshi myopathy; limb girdle muscular dystrophy type 2B (LGMD2B); and distal myopathy. These conditions are collectively known as dysferlinopathies and are caused by more than 600 mutations that have been identified across the DYSF gene to date. In this review, we discuss the key molecular and clinical features of LGMD2B, the causative gene DYSF, and the associated dysferlin protein structure. We also provide an update on current approaches to LGMD2B diagnosis and advances in drug development, including splice switching antisense oligonucleotides. We give a brief update on clinical trials involving adeno-associated viral gene therapy and the current progress on CRISPR/Cas9 mediated therapy for LGMD2B, and then conclude by discussing the prospects of antisense oligomer-based intervention to treat selected mutations causing dysferlinopathies.

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.

Background
Midkine is a multi-functional growth factor/cytokine that is involved in diverse solid and haematological cancers. Midkine mediates critical cell interactions within the tumour microenvironment, and thereby contributes to metastasis (Nature 2017), immunosuppression and resistance to immune checkpoint inhibitors (Nature Medicine 2020; Nature Comms 2022) and angiogenesis. Therefore, midkine may be a novel target, particularly in hard to treat or resistant tumours. While various biologicals inhibit midkine in animal models of cancer, splice switching antisense oligonucleotides (SSOs) have not been evaluated. SSOs uniquely reduce the levels of full length midkine protein and also generate non-functional, truncated midkine due to exon skipping.

Methods
Guided by SpliceAid to identify splice enhancer binding motifs SSOs were designed to predicted splice motifs in exons 3 and 4 of the midkine mRNA and synthesized using 2’OMe-PS nucleotide chemistry. Exon skipping was initially assessed by RT-PCR with primers flanking exons 2 and 5.

Results
Transfection into midkine-expressing human Huh7 liver and SHSY5Y neuroblastoma cancer cells elicited up to 30% of mRNA missing the targeted exons. Optimisation of lead SSOs through microwalking, cocktails of SSOs and PMO chemistry resulted in >90% exon skipped midkine. Importantly, the truncated midkine protein, corresponding to the shorter mRNA lacking Exon 4, was produced by cancer cells. Studies are underway to examine the functional outcomes of midkine SSOs on cancer cell proliferation/apoptosis, migration, invasion and angiogenesis.

Conclusions
Lead midkine SSOs will then be assessed for their ability to alter in vivo tumour growth and metastasis as a prelude for further pre-clinical development.

The pre-mRNA splicing process is an essential aspect of gene expression and function and plays a substantial role in the complexity of higher eukaryotes. The development of antisense oligonucleotides (AOs) to harness the splicing process and manipulate it to treat various inherited and acquired diseases has been boosted by its flexibility and customisation capability. As the amount of research in this space increases, certain aspects need to be considered, in particular, how nonsequential splicing of pre-mRNA can impact AO-mediated splicing manipulation. In this chapter, we reviewed literature discussing intron removal order and several examples of disease-causing mutations impacted by this phenomenon. We also compared two strategies used to study intron removal order and the occasions that they are best suited. Finally, we discuss how nonsequential splicing could facilitate or impede the development of splice-manipulating AOs and aspects to consider when analysing AO effectiveness.