Kelly Martinovich

Introduction: Severity and disease progression in people with Cystic Fibrosis (CF) is typically dependent on their genotype. One potential therapeutic strategy for people with specific mutations is exon skipping with antisense oligonucleotides (AO). CFTR exon 9 is an in-frame exon and hence the exclusion of this exon would excise only 31 amino acids but not alter the reading frame of the remaining mRNA. Splice mutations 1209 + 1 G > C and 1209 + 2 T > G were documented to cause CFTR exon 9 skipping and these variants were reported to manifest as a milder CF disease, therefore exon 9 skipping could be beneficial for people with class I mutations that affect exon 9 such as p.Trp401X. While the impact of exon 9 skipping on gene expression and cellular pathways can be studied in cells in vitro, trace amount of full-length normal or mutated material could confound the evaluation. To overcome this limitation, the impact of CFTR exon 9 skipping on disease phenotype and severity is more effectively evaluated in a small animal model. It was hypothesised that antisense oligonucleotide-mediated skipping this particular exon could result in a “mild mouse CF phenotype”. Methods: Cftr exon 9 deleted mice were generated using homologous recombination. Survival of homozygous (CftrΔ9/Δ9) and heterozygous (CftrΔ9/+) mice was compared to that of other CF mouse models, and lung and intestinal organ histology examined for any pathologies. Primary airway epithelial cells (pAECs) were harvested from CftrΔ9/Δ9 mice and cultured at the Air Liquid Interface for CFTR functional assessment using Ussing Chamber analysis. Results: A CftrΔ9/Δ9 mouse model presented with intestinal obstructions, and at time of weaning (21 days). CftrΔ9/Δ9 mice had a survival rate of 83% that dropped to 38% by day 50. Histological sections of the small intestine from CftrΔ9/Δ9 mice showed more goblet cells and mucus accumulation than samples from the CftrΔ9/+ littermates. Airway epithelial cell cultures established from CftrΔ9/Δ9 mice were not responsive to forskolin stimulation. Summary: The effect of Cftr exon 9 deletion on Cftr function was assessed and it was determined that the encoded Cftr isoform did not result in a milder “mouse CF disease phenotype,” suggesting that Cftr exon 9 is not dispensable, although further investigation in human CF pAECs would be required to confirm this observation.

Current CFTR modulator therapies for cystic fibrosis (CF) act upon the CFTR protein to improve conformation and function. Although 90% of people with CF have a genotype that can benefit from these treatments, some of the remaining 10% have nonsense or frameshift mutations that result in premature termination of protein translation and nonsense mediated decay (NMD). These mutations result in no CFTR protein being produced, and therefore no benefit will be derived from CFTR modulating drugs. One potential complementary therapeutic strategy is antisense oligonucleotide (AO) mediated splice modulation. The AOs are synthetic RNA analogues designed to anneal to selected splice motifs within the pre-mRNA. The binding of an AO alters the recognition of the splice site or motif by the spliceosome and therefore modulates exon selection. We hypothesize that skipping of selected exons using AOs in people with CF caused by NMD-prone mutations such as p.Glu92X(exon 4), p.Tyr275X(exon 7), p.Phe861Leufsx3(exon 15) or c.2989-1G>A(exon 18) the premature termination of translation can be avoided. The protein isoform generated from the induced RNA isoform may become amenable to CFTR modulators.

Introduction/Aim. Over 2000 different mutations have been reported in patients with Cystic Fibrosis (CF) and found to occur in all CFTR exons and introns. Many mutations are not amenable to current therapies, and therefore new drugs must be developed. Antisense oligonucleotides (AOs) are synthetic nucleic acid analogues designed to anneal to selected splice motifs within pre-mRNAs. AO binding alters the recognition of the splice site by the spliceosome and therefore modulates exon selection. c.2989-1G > A causes skipping of CFTR exon 19, which disrupts the reading frame, and abolishes CFTR protein production. We hypothesize by also skipping exon 18 in patients with c.2989-1G > A the reading frame will be restored, and the induced protein isoform may regain function or become amenable to CFTR modifying drugs.

Methods. Three AO sequences were initially optimised using 2’-OMethyl modified bases on a phosphorothioate backbone (2OMe) and transfected using lipofectamine into primary airway epithelial cells (AECs) from non-CF and child with CF causing c.2989-1G > A/ c.2989-1G > A. The transfection is left for 48 hours after which the cells are collected, and RNA extracted. PCR primers that amplify CFTR exons 17 to 21 are used to determine the efficacy of exon skipping. PCR bands are measured using densitometry and the density of the full-length band to the exon skipped band is compared.

Results. The 2OMe sequence produced 64% exon 18 skipping in c.2989-1G > A/ c.2989-1G > A CF airway epithelial cells. In non-CF AECs the efficiency was greatly reduced to only 11% exon 18 skipping.

Conclusion. Exon 18 can be efficiently skipped from the CFTR transcript in c.2989-1G > A/ c.2989-1G > A CF-derived airway epithelial cells. We propose that exon skipping to restore the reading frame of the CFTR gene, resulting in improved CFTR protein production. This new internally truncated CFTR protein could have improve function and/or become amenable to CFTR modifying drugs.

Introduction/Aim: Children with Cystic Fibrosis (CF) typically exhibit prolonged and severe symptoms during rhinovirus (RV) infection compared to healthy children. Here, we studied the host-rhinovirus interaction signature after infection, integrating two omics: (1) Whole transcriptome sequencing (WTS) to determine the viral load and host’s gene expression, and (2) metabolomics to profile the metabolites associated with the viral infection of primary tracheal epithelial cells obtained from healthy children (H) and those with CF. Methods: WTS and hydrophilic interaction liquid chromatography (HILIC) coupled to mass spectrometry, were used to identify the differences in transcripts/metabolites and RV coverage produced by H (3.9 ± 1.5 years; n=8) and CF (2.6 ± 1.8 years; n=8; all p.Phe508del/p.Phe508del) primary epithelial cells at 24 hours post-infection with human rhinovirus 1B. Univariate and multivariate analyses were then performed to identify the infection hallmark. Results: RV coverage in uninfected cells (Mock) was less than 0.5X, whereas, infected cells presented 44.4X and 101.6X of RV in children with and without CF respectively. Global RV infection was associated with 14 metabolites and 1713 genes differential expressed. From these, several metabolic pathways were found dysregulated including inositol metabolism and glycerophospholipid biosynthesis. Conclusion: Metabolic host-derived pathways associated with RV infection were identified. Although functional analysis is still required, these pathways could be used as potential biomarkers of RV infection in CF. Future analysis will help to understand whether these compounds can be targeted for antiviral purposes.

Introduction: Over 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene causes cystic fibrosis (CF) with variable clinical phenotypes. The length of the poly T tract in intron 9 influences exon 10 selection and can manifest as mild or severe disease depending on other CFTR mutations. Manipulation of CFTR pre-RNA splicing using antisense oligonucleotides (AOs) is a potential therapy for selected CF-causing mutations. We aim to develop splice modulating AOs 2019 Cystic Fibrosis Conference 218 to rescue CFTR function in CF patients that carry the shorter 5T polymorphism in intron 9. AOs could strengthen exon 10 selection or weaken the selection of flanking exons. As seen with specific cases of Duchenne muscular dystrophy, removing a block of exons can restore more functional dystrophin protein over the removal of a single exon.

Methods: Multiple AOs targeting CFTR intron 9 and the flanking exons; 9 and 11 were designed and initially optimised using 2’-O-methyl modified bases on a phosphorothioate backbone (2OMe) and transfected into primary airway epithelial cells from a child with p.508del/ Arg117His;5T CF. After 48 hours RNA was collected, and PCR was used to determine the ratio of altered transcript compared to full-length product. CFTR protein size was determined by Western blot analysis. CFTR functional outcomes were measured using Ussing chamber studies utilising air-liquid interface primary airway cell cultures.

Results: Of the 32 2OMe AOs tested for exon 10 inclusion, none reduced the intron 9 5T induced exon 10 skipping. Of the 8 AOs designed to skip exon 9, the highest efficiency was 24% from both the p.Phe508del allele and intron 9 5T allele. Of the 6 AOs designed to skip Exon 11, the highest efficiency was 22% from the intron 9 5T allele. CFTR protein size was determined on Western blot and CFTR function was determined by response to forskolin (change in Isc).

Conclusion: We propose that skipping the exons flanking exon 10 (9 and/or 11) on the CFTR 5T allele could improve CFTR function in CF patients carrying selected mutations, either alone or in combination with current therapeutics.

Acknowledgments: Supported by USCF; NHMRC; CFWA.

Introduction: Over 2000 different mutations have been reported in patients with CF and found to occur in all CFTR exons and introns. Of these, 168 are nonsense mutations, 295 are frameshift mutations that are not amenable to current therapies, and therefore new drugs must be developed. Antisense oligonucleotides (AOs) are synthetic RNA analogues that can be designed to anneal to selected splice motifs within pre-mRNAs. AO binding alters the recognition of the splice site by the spliceosome and therefore modulates exon selection. Exon 15 has been selected as an initial target since it has been reported to harbour ~40 mutations and exclusion of this exon will not disrupt the mRNA reading frame. We hypothesize that by skipping exon 15 in patients with amenable mutations such as p.Phe861Leufsx3, studied here, the disease-causing mutation can be bypassed and the induced isoform may retain some residual function, therefore altering the course of disease.

Methods: AO sequences were initially optimised using 2’-O-methyl modified bases on a phosphorothioate backbone (2OMe) and transfected into monolayer primary non-CF (2.6 years M) and p.Phe861Leufsx3/p. Phe508del CF airway epithelial cells (4.1 years M). The ratio of the AO induced, CFTR RT-PCR transcript product missing the target exon, relative to the full length product provides an estimate of AO exon skipping efficiency. The most effective 2OMe AO sequence was identified and re-synthesised as the clinically validated phosphorodiamidate morpholino (PMO) chemistry. Monolayer transfections were repeated. AO mediated modification of protein was shown by Western blot analysis and comparison to size standards. CFTR function before and after PMO application was measured using Ussing chamber studies.

Results: 2OMe AOs were designed, evaluated and further optimised by micro-walking around sequences shown to be capable of modifying splicing. The 2OMe sequence that was most efficient induced an estimated 50% skipping in p.Phe861Leufsx3/p.Phe508del CF airway epithelial cells The PMO induced efficient skipping of exon 15 from non-CF (49%) and p.Phe861Leufsx3/p.Phe508del CF (88%) cells after 7 days in culture. Western blot was used to determine the effect on the induced CFTR protein. Airway epithelial cells from children with CF were also grown at air-liquid interface 28 days to become mucocillary differentiated then assessed for CFTR function using an Ussing chamber.

Conclusion: Exon 15 can be efficiently skipped from the CFTR transcript in both non-CF and CF-derived airway epithelial cells. We propose that exon skipping to remove disease-causing mutations in selected in-frame exons can improve function in amenable CF patients, either alone or in combination with current therapeutics.

Acknowledgments: Supported by USCF; NHMRC; CFWA.

Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations result in cystic fibrosis (CF) disease and a broad spectrum of clinical phenotypes are observed in CF patients. One mutation, Arg117His affects the conductivity of the CFTR channel and can result in a mild or severe phenotype, influenced by co‐location of the mutation and an intron 9 polymorphism: a poly T tract varying from 5 to 9 nucleotid. The shorter ‘5T’ allele weakens the intron 9 acceptor site and promotes exclusion of exon 10 from the mature CFTR transcript, resulting in a non‐functional CFTR channel, and a more severe disease phenotyp. Manipulation of CFTR pre‐RNA splicing using antisense oligonucleotides (AOs) is a potential therapy for those CF patients with this particular mutation. This study explores antisense therapy to correct abnormal splicing of CFTR RNA and improve CFTR function. AOs targeting CFTR intron 9, around the specific 5T polymorphism, were designed and transfected into monolayer primary airway epithelial cells from a CF patient harbouring this disease‐associated polymorphism. AOs that decrease the levels of CFTR transcript missing exon 10 will then be assessed for CFTR function using an Ussing Chamber and CF patient primary epithelial cells grown at the Air‐liquid interface. We propose that corrected splicing of the CFTR 5T allele will improve function in CF patients carrying selected mutations, either alone or in combination with current therapeutics.

Antisense oligonucleotides are an emerging therapeutic option to treat diseases with known genetic origin. In the age of personalised medicines, antisense oligonucleotides can sometimes be designed to target and bypass or overcome a patient’s genetic mutation, in particular those lesions that compromise normal pre-mRNA processing. Antisense oligonucleotides can alter gene expression through a variety of mechanisms as determined by the chemistry and antisense oligomer design. Through targeting the pre-mRNA, antisense oligonucleotides can alter splicing and induce a specific spliceoform or disrupt the reading frame, target an RNA transcript for degradation through RNaseH activation, block ribosome initiation of protein translation or disrupt miRNA function. The recent accelerated approval of eteplirsen (renamed Exondys 51™) by the Food and Drug Administration, for the treatment of Duchenne muscular dystrophy, and nusinersen, for the treatment of spinal muscular atrophy, herald a new and exciting era in splice-switching antisense oligonucleotide applications to treat inherited diseases. This review considers the potential of antisense oligonucleotides to treat inherited lung diseases of childhood with a focus on cystic fibrosis and disorders of surfactant protein metabolism.