Chalermchai Mitrpant

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.

Spinal muscular atrophy (SMA) is a severe, debilitating neuromuscular condition characterised by loss of motor neurons and progressive muscle wasting. SMA is caused by a loss of expression of SMN1 that encodes the survival motor neuron (SMN) protein necessary for the survival of motor neurons. Restoration of SMN expression through increased inclusion of SMN2 exon 7 is known to ameliorate symptoms in SMA patients. As a consequence, regulation of pre-mRNA splicing of SMN2 could provide a potential molecular therapy for SMA. In this study, we explored if splice switching antisense oligonucleotides could redirect the splicing repressor hnRNPA1 to the hnRNPA1b isoform and restore SMN expression in fibroblasts from a type I SMA patient. Antisense oligonucleotides (AOs) were designed to promote exon 7b retention in the mature mRNA and induce the hnRNPA1b isoform. RT-PCR and western blot analysis were used to assess and monitor the efficiency of different AO combinations. A combination of AOs targeting multiple silencing motifs in hnRNPA1 pre-mRNA led to robust hnRNPA1b induction, which, in turn, significantly increased expression of full-length SMN (FL-SMN) protein. A combination of PMOs targeting the same motifs also strongly induced hnRNPA1b isoform, but surprisingly SMN2 exon 5 skipping was detected, and the PMO cocktail did not lead to a significant increase in expression of FL-SMN protein. We further performed RNA sequencing to assess the genome-wide effects of hnRNPA1b induction. Some 3244 genes were differentially expressed between the hnRNPA1b-induced and untreated SMA fibroblasts, which are functionally enriched in cell cycle and chromosome segregation processes. RT-PCR analysis demonstrated that expression of the master regulator of these enrichment pathways, MYBL2 and FOXM1B, were reduced in response to PMO treatment. These findings suggested that induction of hnRNPA1b can promote SMN protein expression, but not at sufficient levels to be clinically relevant.

Mutations in the USH2A gene are the most common cause of Usher syndrome and autosomal recessive non-syndromic retinitis pigmentosa. Here, we describe the generation of three induced pluripotent stem cell lines from dermal fibroblasts derived from a patient carrying biallelic c.949C > A and c.1256G > T variants in the USH2A gene, using episomal reprogramming plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28, mir302/367 and shRNA targeting TP53. All three lines expressed pluripotency markers, displayed unaltered karyotypes as well as trilineage differentiation potential, and were negative for reprogramming episomes and mycoplasma.

Purpose : Retinitis pigmentosa 11 (RP11) is an inherited degenerative retinal disease caused by heterozygous mutations in pre-mRNA processing factor 31 (PRPF31) for which there is currently no effective treatment available. RP11 features incomplete penetrance within affected families; the level of PRPF31 expression from the healthy allele determines whether mutation carriers develop symptoms. The CCR4-NOT transcription complex subunit 3 (CNOT3) is a major disease modifier that regulates PRPF31 levels via transcription inhibition. Higher CNOT3 levels are observed in RP11 cases. In this study, we aim to lower CNOT3 expression and function to indirectly upregulate functional PRPF31 from the healthy allele and rescue RP11 disease phenotypes. Methods : Seventy-four antisense oligomers (ASO) were designed to target exonic splice enhancers to mediate exclusion of selected CNOT3 exons to (i) induce translational frameshift and mRNA decay or (ii) produce truncated low/non-functional CNOT3 isoform(s). PRPF31 expression and function were assessed in RP11 iPSC-derived retinal pigment epithelial (RPE) cells. Results : We observed an inverse correlation between CNOT3 and PRPF31 mRNA levels in healthy fibroblasts (n=20). In iPSC-RPE cells, we found 10% higher expression of CNOT3 with 17% lower PRPF31 expression in a patient compared to an asymptomatic relative, both carrying a PRPF31 c.1205 C>A nonsense mutation. Fewer and shorter primary cilia were observed in the symptomatic patient RPE compared to asymptomatic, healthy RPE. Lowering CNOT3 levels and function with ASOs demonstrated a 1.7-fold increase in PRPF31 expression in patient RPE and significantly improved cilia number and length. Healthy cilia play an integral role in phagocytosis and are crucial for normal retinal function. Conclusions : Subtle changes in CNOT3 and PRPF31 levels in retinal cells determine disease penetrance in PRPF31 mutation carriers within an affected family. Modulating expression levels of these proteins can reverse the cellular disease phenotype in RP11. ASOs are effective modulators of CNOT3 expression and function with the ability to increase PRPF31 transcription from the unaffected allele to an expected therapeutic level. Future studies will assess the restoration of transcriptional profiles linked to improved PRPF31 levels in patient derived iPSC-retinal organoids and RPE compared to healthy controls.

The literature surrounding the use of antisense oligonucleotides continues to grow, with new disease and mechanistic applications constantly evolving. Furthermore, the discovery and advancement of novel chemistries continues to improve antisense delivery, stability and effectiveness. For each new application, a rational sequence design is recommended for each oligomer, as is chemistry and delivery optimization. To confirm oligomer delivery and antisense activity, a positive control AO sequence with well characterized target-specific effects is recommended. Here, we describe splice-switching antisense oligomer sequences targeting the ubiquitously expressed human and mouse SMN and Smn genes for use as control AOs for this purpose. We report two AO sequences that induce targeted skipping of SMN1/SMN2 exon 7 and two sequences targeting the Smn gene, that induce skipping of exon 5 and exon 7. These antisense sequences proved effective in inducing alternative splicing in both in vitro and in vivo models and are therefore broadly applicable as controls. Not surprisingly, we discovered a number of differences in efficiency of exon removal between the two species, further highlighting the differences in splice regulation between species.

Antisense oligonucleotides are short, single-stranded nucleic acid analogues that can interfere with pre-messenger RNA (pre-mRNA) processing and induce excision of a targeted exon from the mature transcript. When developing a panel of antisense oligonucleotides to skip every dystrophin exon, we found great variation in splice switching efficiencies, with some antisense oligonucleotides ineffective, even when directed to canonical splice sites and transfected into cells at high concentrations. In this study, we re-evaluated some of these ineffective antisense oligonucleotide sequences after incorporation of locked nucleic acid residues to increase annealing potential. Antisense oligonucleotides targeting exons 16, 23, and 51 of human DMD transcripts were synthesized as two different chemistries, 2 '-O-methyl modified bases on a phosphorothioate backbone or mixmers containing several locked nucleic acid residues, which were then transfected into primary human myotubes, and DMD transcripts were analyzed for exon skipping. The ineffective 2 '-O-methyl modified antisense oligonucleotides induced no detectable exon skipping, while all corresponding mixmers did induce excision of the targeted exons. Interestingly, the mixmer targeting exon 51 induced two unexpected transcripts arising from partial skipping of exon 51 with retention of 95 or 188 bases from the 5 ' region of exon 51. These results indicated that locked nucleic acid/2 '-O-methyl mixmers are more effective at inducing exon skipping, however, this improvement may come at the cost of activating alternative cryptic splice sites and off-target effects on gene expression.

The severe childhood disease spinal muscular atrophy (SMA) arises from the homozygous loss of the survival motor neuron 1 gene (SMN1). A homologous gene potentially encoding an identical protein, SMN2 can partially compensate for the loss of SMN1, however the exclusion of a critical exon in the coding region during mRNA maturation results in insufficient levels of functional protein. The rate of transcription is known to influence the alternative splicing of gene transcripts, with a fast transcription rate correlating to an increase in alternative splicing. Conversely, a slower transcription rate is more likely to result in the inclusion of all exons in the transcript. Targeting SMN2 with antisense oligonucleotides to influence the processing of terminal exon 8 could be a way to slow transcription and induce the inclusion of exon 7. Interestingly, following oligomer treatment of SMA patient fibroblasts, we observed the inclusion of exon 7 as well as intron 7 in the transcript. Since the normal termination codon is located in exon 7, this exon/intron7-SMN2 transcript should encode the normal protein, and only carry a longer 3´ untranslated region. Further studies showed the extra 3´UTR length contained a number of regulatory motifs that modify transcript and protein regulation, leading to translational repression of SMN. While unlikely to provide therapeutic benefit for spinal muscular atrophy patients, this novel technique for gene regulation could provide another avenue for the repression of undesirable gene expression in a variety of other diseases.

We sought to use splice-switching antisense oligonucleotides to produce a model of accelerated ageing by enhancing expression of progerin, translated from a mis-spliced lamin A gene ( LMNA) transcript in human myogenic cells. The progerin transcript (LMNA Δ150) lacks the last 150 bases of exon 11, and is translated into a truncated protein associated with the severe premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS). HGPS arises from de novo mutations that activate a cryptic splice site in exon 11 of LMNA and result in progerin accumulation in tissues of mesodermal origin. Progerin has also been proposed to play a role in the 'natural' ageing process in tissues. We sought to test this hypothesis by producing a model of accelerated muscle ageing in human myogenic cells. A panel of splice-switching antisense oligonucleotides were designed to anneal across exon 11 of the LMNA pre-mRNA, and these compounds were transfected into primary human myogenic cells. RT-PCR showed that the majority of oligonucleotides were able to modify LMNA transcript processing. Oligonucleotides that annealed within the 150 base region of exon 11 that is missing in the progerin transcript, as well as those that targeted the normal exon 11 donor site induced the LMNA Δ150 transcript, but most oligonucleotides also generated variable levels of LMNA transcript missing the entire exon 11. Upon evaluation of different oligomer chemistries, the morpholino phosphorodiamidate oligonucleotides were found to be more efficient than the equivalent sequences prepared as oligonucleotides with 2′-O-methyl modified bases on a phosphorothioate backbone. The morpholino oligonucleotides induced nuclear localised progerin, demonstrated by immunostaining, and morphological nuclear changes typical of HGPS cells. We show that it is possible to induce progerin expression in myogenic cells using splice-switching oligonucleotides to redirect splicing of LMNA. This may offer a model to investigate the role of progerin in premature muscle ageing.

In the search for the most efficacious antisense oligonucleotides (AOs) aimed at inducing SMN2 exon 7 inclusion, we systematically assessed three AOs, PMO25 (−10, −34), PMO18 (−10, −27), and PMO20 (−10, −29), complementary to the SMN2 intron 7 splicing silencer (ISS-N1). PMO25 was the most efficacious in augmenting exon 7 inclusion in vitro in spinal muscular atrophy (SMA) patient fibroblasts and in vitro splicing assays. PMO25 and PMO18 were compared further in a mouse model of severe SMA. After a single intracerebroventricular (ICV) injection in neonatal mice, PMO25 increased the life span of severe SMA mice up to 30-fold, with average survival greater by 3-fold compared with PMO18 at a dose of 20 μg/g and 2-fold at 40 μg/g. Exon 7 inclusion was increased in the CNS but not in peripheral tissues. Systemic delivery of PMO25 at birth achieved a similar outcome and produced increased exon 7 inclusion both in the CNS and peripherally. Systemic administration of a 10-μg/g concentration of PMO25 conjugated to an octaguanidine dendrimer (VMO25) increased the life span only 2-fold in neonatal type I SMA mice, although it prevented tail necrosis in mild SMA mice. Higher doses and ICV injection of VMO25 were associated with toxicity. We conclude that (1) the 25-mer AO is more efficient than the 18-mer and 20-mer in modifying SMN2 splicing in vitro; (2) it is more efficient in prolonging survival in SMA mice; and (3) naked Morpholino oligomers are more efficient and safer than the Vivo-Morpholino and have potential for future SMA clinical applications.

Age-related changes in splice-forms of LMNA, which encodes the nuclear lamina proteins lamin A/C, have not been investigated in skeletal muscle. In the rare premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS), de novo point mutations in LMNA activate a cryptic splice site in exon 11, resulting in a 150 base deletion in LMNA mRNA and accumulation of a truncated protein isoform, progerin. The LMNA ∆150 progerin transcript has also been found in trace quantities in tissues of healthy people and its implication in ‘natural’ ageing has been proposed. We therefore investigated the expression of progerin and lamin A/C in normal human and mouse skeletal muscles of different ages. LMNA ∆150 was detected in most muscle samples from healthy individuals aged 16-71 years, but was not present in any mouse muscle samples up to the age of 18 months. Real time qPCR of human muscle samples showed that there was an age-related increase in both the full length lamin A and LMNA ∆150 transcripts, whereas their protein levels did not change significantly with age. These findings indicate that there is a basal level of mis-splicing during LMNA expression that does not change with ageing in human muscle, but at levels that do not result in increased aberrant protein. The significance of these findings in the pathophysiology of muscle ageing is uncertain and warrants further investigation.