Jessica Cale

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.

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.

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 type-1 fibrillinopathies are a family of connective tissue disorders of which Marfan syndrome is the most common, affecting between 2-3 in 10,000 individuals. Marfan syndrome is a multisystem disorder characterised by ocular, skeletal and cardiovascular abnormalities and can be caused by any one of over 2800 unique mutations reported across the fibrillin-1 (FBN1) gene. FBN1 encodes the large extracellular glycoprotein, fibrillin-1; the fibrillin-1 monomers aggregate to form the backbone of microfibrils. Fibrillin-1 has both structural and regulatory roles, including the regulation of transforming growth factor-beta. This regulation is critical in maintaining extracellular matrix stability and dysregulation of this function is one of the keystones of the Marfan syndrome pathogenesis. Mutations in FBN1 can result in reduced fibrillin-1 expression, loss-of-function or the production of two different fibrillin-1 proteins that are unable to interact to form functional microfibrils. The result in all three cases is a lack of functional microfibrils and destabilisation of the extracellular matrix. The current standard of care relies heavily on surgical intervention and lifelong use of medications to slow disease progression, thus the need for new therapeutic options that target the cause of disease. This thesis focused on developing a suite of short synthetic nucleic acid sequences, known as antisense oligonucleotides, to selectively manipulate FBN1 pre-mRNA splicing. We hypothesised that the removal of an amenable mutation-associated exon would result in one of the following scenarios. For missense mutations, removing the mutation-associated exon from affected and unaffected transcripts would eliminate the aberrant sequence and restore homogeneity between fibrillin-1 monomers. For splice-site and in-frame deletion mutations, excluding the mutation-associated exon from the remaining healthy transcripts would restore the domain periodicity and monomer homogeneity. Lastly, for mutations resulting in a premature termination codon, excluding the mutation-associated exon from the affected transcripts would restore the reading frame, rescuing transcript functionality. The mutation-associated exon would also need to be removed from the unaffected transcripts to maintain monomer homogeneity. For each of these scenarios, we hypothesised that the internally truncated proteins produced would be capable of forming functional microfibrils, thereby reducing the severity or slowing the progression of the Marfan syndrome phenotype. As an initial proof-of-concept for this project, antisense oligonucleotide sequences targeting FBN1 exon 52 were assessed. A promising sequence induced dose-dependent exon skipping in healthy control cells allowing us to observe the formation of healthy fibrillin-1 fibres with 0% exon skipping, loss of extruded fibrillin-1 fibres with 50% skipping; mimicking the disease-like state, and subsequent re-appearance of extracellular fibrillin-1 fibres with greater than 80% skipping indicating that the internally truncated fibrillin-1 monomers are capable of forming aggregates. Similarly, we demonstrate that FBN1 exons 47 and 59 can be efficiently excluded, and sufficient skipping can result in fibrillin-1 fibre formation. However, many of the FBN1 exons targeted were not as readily excised from the mature mRNA. Comparison of three antisense oligonucleotide chemistries revealed the promising efficacy of the newer thiophosphoramidate morpholino oligomer chemistry. Similar to the commonly used phosphorodiamidate morpholino oligomer, the thiophosphoramidate morpholino oligomer sequences resulted in efficient and consistent FBN1 exon 52 skipping. Both chemistries also had little effect on paraspeckle protein distribution, an indicator of toxicity, unlike the third, 2′OMe-PS, chemistry that caused gross paraspeckle protein disruption. Therefore, thiophosphoramidate morpholino oligomer should be included in the repertoire of chemistries routinely used in studies developing antisense therapeutics. Lastly, while we demonstrate that >80% exon skipping can lead to fibrillin-1 microfibril-like formations in vitro, we could not confirm the functionality of these fibres nor the effect of exon skipping on the Marfan syndrome phenotype. Nevertheless, this study demonstrates proof-of-concept and lays a solid foundation for further development of antisense oligonucleotides to treat the type-1 fibrillinopathies.

Marfan syndrome is one of the most common dominantly inherited connective tissue disorders, affecting 2–3 in 10,000 individuals, and is caused by one of over 2800 unique FBN1 mutations. Mutations in FBN1 result in reduced fibrillin-1 expression, or the production of two different fibrillin-1 monomers unable to interact to form functional microfibrils. Here, we describe in vitro evaluation of antisense oligonucleotides designed to mediate exclusion of FBN1 exon 52 during pre-mRNA splicing to restore monomer homology. Antisense oligonucleotide sequences were screened in healthy control fibroblasts. The most effective sequence was synthesised as a phosphorodiamidate morpholino oligomer, a chemistry shown to be safe and effective clinically. We show that exon 52 can be excluded in up to 100% of FBN1 transcripts in healthy control fibroblasts transfected with PMO52. Immunofluorescent staining revealed the loss of fibrillin 1 fibres with ~50% skipping and the subsequent re-appearance of fibres with >80% skipping. However, the effect of exon skipping on the function of the induced fibrillin-1 isoform remains to be explored. Therefore, these findings demonstrate proof-of-concept that exclusion of an exon from FBN1 pre-mRNA can result in internally truncated but identical monomers capable of forming fibres and lay a foundation for further investigation to determine the effect of exon skipping on fibrillin-1 function.

Background: Rare diseases are chronic and debilitating and while individually they affect less than 1 in 2,000 people, collectively they have a huge impact affect around 1.8 million Australians. Approximately 80% of these have a genetic origin, however only 30% of patients receive a formal molecular diagnosis. RASopathies are a group of rare disease that are caused by a mutations in the genes involved in the RAS-MAPK pathway, the most common of which is Noonan syndrome, which is characterised by heart defects, short stature, chest deformities, and specific craniofacial features. This study focuses on the effects of a novel c.173C>T (p.Thr58Ile) mutation, found in the NRAS gene of a patient diagnosed with Noonan-like syndrome, on the localisation and function of the NRAS protein. In doing so this study aimed to validate the role of mutations in the patient’s disease and provide information toward the creation of an experimental pipeline for the validation of other rare variants. Methods: U87-MG cells were transiently transfected with the NRAS constructs tagged with GFP2, and stained with specific antibody markers to determine localisation of the proteins using confocal microscopy. Functional studies included an Annexin V apoptosis assay, using flow cytometry to detect and quantify levels of apoptosis in transfected and untransfected populations, and the prediction of conserved domains using online bioinformatic tools. Results and Conclusions: Preliminary results from this study suggest that there is a difference between the localisation and function of the mutant and wild type proteins. The mutant protein was seen to co-localise with the Golgi apparatus as expected if the mutant protein was constitutively active. However the mutant protein was also observed to co-localise with the markers for the plasma membrane, nucleus and endoplasmic reticulum indicating that the mutation affects more than just the activation of the protein. An increase in apoptosis observed in NRASMUT transfected population, in comparison to both the wild type and untransfected populations, indicates that the mutation is engaging the pro-apoptotic function of NRAS associated with an increased activation of the RAS–RAF–MAPKK–MAPK pathway. Bioinformatic analyses identified that the mutation is location within a number of motifs involved in the binding of GTP and thus the activation and inactivation of NRAS, directing further studies toward the proliferation, activation and interaction of NRAS with effectors.