Limb Girdle Muscular Dystrophy Type 2B (LGMD2B): Diagnosis and Therapeutic Possibilities.

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.

A Precision Therapy Approach for Retinitis Pigmentosa 11 Using Splice-Switching Antisense Oligonucleotides to Restore the Open Reading Frame of PRPF31.

Retinitis pigmentosa 11 is an untreatable, dominantly inherited retinal disease caused by heterozygous mutations in pre-mRNA processing factor 31 PRPF31. The expression level of PRPF31 is linked to incomplete penetrance in affected families; mutation carriers with higher PRPF31 expression can remain asymptomatic. The current study explores an antisense oligonucleotide exon skipping strategy to treat RP11 caused by truncating mutations within PRPF31 exon 12 since it does not appear to encode any domains essential for PRPF31 protein function. Cells derived from a patient carrying a PRPF31 1205C>A nonsense mutation were investigated; PRPF31 transcripts encoded by the 1205C>A allele were undetectable due to nonsense-mediated mRNA decay, resulting in a 46% reduction in PRPF31 mRNA, relative to healthy donor cells. Antisense oligonucleotide-induced skipping of exon 12 rescued the open reading frame with consequent 1.7-fold PRPF31 mRNA upregulation in the RP11 patient fibroblasts. The level of PRPF31 upregulation met the predicted therapeutic threshold of expression inferred in a non-penetrant carrier family member harbouring the same mutation. This study demonstrated increased PRPF31 expression and retention of the nuclear translocation capability for the induced PRPF31 isoform. Future studies should evaluate the function of the induced PRPF31 protein on pre-mRNA splicing in retinal cells to validate the therapeutic approach for amenable RP11-causing mutations.

Characterising splicing defects of ABCA4 variants within exons 13-50 in patient-derived fibroblasts.

The ATP-binding cassette subfamily A member 4 gene (ABCA4)-associated retinopathy, Stargardt disease, is the most common monogenic inherited retinal disease. Given the pathogenicity of numerous ABCA4 variants is yet to be examined and a significant proportion (more than 15%) of ABCA4 variants are categorized as splice variants in silico, we therefore established a fibroblast-based splice assay to analyze ABCA4 variants in an Australian Stargardt disease cohort and characterize the pathogenic mechanisms of ABCA4 variants. A cohort of 67 patients clinically diagnosed with Stargardt disease was recruited. Genomic DNA was analysed using a commercial panel for ABCA4 variant detection and the consequences of ABCA4 variants were predicted in silico. Dermal fibroblasts were propagated from skin biopsies, total RNA was extracted and the ABCA4 transcript was amplified by RT-PCR. Our analysis identified a total of 67 unique alleles carrying 74 unique variants. The most prevalent splice-affecting complex allele c.[5461-10T>C; 5603A>T] was carried by 10% of patients in a compound heterozygous state. ABCA4 transcripts from exon 13 to exon 50 were readily detected in fibroblasts. In this region, aberrant splicing was evident in 10 out of 57 variant transcripts (18%), carried by 19 patients (28%). Patient-derived fibroblasts provide a feasible platform for identification of ABCA4 splice variants located within exons 13-50. Experimental evidence of aberrant splicing contributes to the pathogenic classification for ABCA4 variants. Moreover, identification of variants that affect splicing processes provides opportunities for intervention, in particular antisense oligonucleotide-mediated splice correction.

NEAT1 polyA-modulating antisense oligonucleotides reveal opposing functions for both long non-coding RNA isoforms in neuroblastoma.

Many long non-coding RNAs (lncRNA) are highly dysregulated in cancer and are emerging as therapeutic targets. One example is NEAT1, which consists of two overlapping lncRNA isoforms, NEAT1_1 (3.7 kb) and NEAT1_2 (23 kb), that are functionally distinct. The longer NEAT1_2 is responsible for scaffolding gene-regulatory nuclear bodies termed paraspeckles, whereas NEAT1_1 is involved in paraspeckle-independent function. The NEAT1 isoform ratio is dependent on the efficient cleavage and polyadenylation of NEAT1_1 at the expense of NEAT1_2. Here, we developed a targeted antisense oligonucleotide (ASO) approach to sterically block NEAT1_1 polyadenylation processing, achieving upregulation of NEAT1_2 and abundant paraspeckles. We have applied these ASOs to cells of the heterogeneous infant cancer, neuroblastoma, as we found higher NEAT1_1:NEAT1_2 ratio and lack of paraspeckles in high-risk neuroblastoma cells. These ASOs decrease NEAT1_1 levels, increase NEAT1_2/paraspeckles and concomitantly reduce cell viability in high-risk neuroblastoma specifically. In contrast, overexpression of NEAT1_1 has the opposite effect, increasing cell proliferation. Transcriptomic analyses of high-risk neuroblastoma cells with altered NEAT1 ratios and increased paraspeckle abundance after ASO treatment showed an upregulation of differentiation pathways, as opposed to the usual aggressive neuroblastic phenotype. Thus, we have developed potential anti-cancer ASO drugs that can transiently increase growth-inhibiting NEAT1_2 RNA at the expense of growth-promoting NEAT1_1 RNA. These ASOs, unlike others that degrade lncRNAs, provide insights into the importance of altering lncRNA polyadenylation events to suppress tumorigenesis as a strategy to combat cancer.

Antisense Oligonucleotide Induction of the hnRNPA1b Isoform Affects Pre-mRNA Splicing of SMN2 in SMA Type I Fibroblasts.

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.

Gene replacement therapy restores RCBTB1 expression and cilium length in patient-derived retinal pigment epithelium.

Biallelic mutations in the RCBTB1 gene cause retinal dystrophy. Here, we characterized the effects of RCBTB1 gene deficiency in retinal pigment epithelial (RPE) cells derived from a patient with RCBTB1-associated retinopathy and restored RCBTB1 expression in these cells using adeno-associated viral (AAV) vectors. Induced pluripotent stem cells derived from a patient with compound heterozygous RCBTB1 mutations (c.170delG and c.707delA) and healthy control subjects were differentiated into RPE cells. RPE cells were treated with AAV vectors carrying a RCBTB1 transgene. Patient-derived RPE cells showed reduced expression of RCBTB1. Expression of NFE2L2 showed a non-significant reduction in patient RPE cells compared with controls, while expression of its target genes (RXRA, IDH1 and SLC25A25) was significantly reduced. Trans-epithelial electrical resistance, surface microvillus densities and primary cilium lengths were reduced in patient-derived RPE cells, compared with controls. Treatment of patient RPE with AAV vectors significantly increased RCBTB1, NFE2L2 and RXRA expression and cilium lengths. Our study provides the first report examining the phenotype of RPE cells derived from a patient with RCBTB1-associated retinopathy. Furthermore, treatment of patient-derived RPE with AAV-RCBTB1 vectors corrected deficits in gene expression and RPE ultrastructure, supporting the use of gene replacement therapy for treating this inherited retinal disease.

Proof-of-Concept: Antisense Oligonucleotide Mediated Skipping of Fibrillin-1 Exon 52.

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.

Progress in the molecular pathogenesis and nucleic acid therapeutics for Parkinson's disease in the precision medicine era.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders that manifest various motor and nonmotor symptoms. Although currently available therapies can alleviate some of the symptoms, the disease continues to progress, leading eventually to severe motor and cognitive decline and reduced life expectancy. The past two decades have witnessed rapid progress in our understanding of the molecular and genetic pathogenesis of the disease, paving the way for the development of new therapeutic approaches to arrest or delay the neurodegenerative process. As a result of these advances, biomarker-driven subtyping is making it possible to stratify PD patients into more homogeneous subgroups that may better respond to potential genetic-molecular pathway targeted disease-modifying therapies. Therapeutic nucleic acid oligomers can bind to target gene sequences with very high specificity in a base-pairing manner and precisely modulate downstream molecular events. Recently, nucleic acid therapeutics have proven effective in the treatment of a number of severe neurological and neuromuscular disorders, drawing increasing attention to the possibility of developing novel molecular therapies for PD. In this review, we update the molecular pathogenesis of PD and discuss progress in the use of antisense oligonucleotides, small interfering RNAs, short hairpin RNAs, aptamers, and microRNA-based therapeutics to target critical elements in the pathogenesis of PD that could have the potential to modify disease progression. In addition, recent advances in the delivery of nucleic acid compounds across the blood-brain barrier and challenges facing PD clinical trials are also reviewed.

Nonsequential Splicing Events Alter Antisense-Mediated Exon Skipping Outcome in COL7A1.

The COL7A1 gene encodes homotrimer fibrils essential for anchoring dermal and epidermal layers, and pathogenic mutations in COL7A1 can cause recessive or dominant dystrophic epidermolysis bullosa. As a monogenic disease gene, COL7A1 constitutes a potential target for antisense oligomer-mediated exon skipping, a therapy applicable to a growing number of other genetic disorders. However, certain characteristics of COL7A1: many exons, low average intron size, and repetitive and guanine-cytosine rich coding sequence, present challenges to the design of specific and effective antisense oligomers. While targeting COL7A1 exons 10 and 73 for excision from the mature mRNA, we discovered that antisense oligomers comprised of 2'-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers produced similar, but distinctive, splicing patterns including excision of adjacent nontargeted exons and/or retention of nearby introns in some transcripts. We found that the nonsequential splicing of certain introns may alter pre-mRNA processing during antisense oligomer-mediated exon skipping and, therefore, additional studies are required to determine if the order of intron removal influences multiexon skipping and/or intron retention in processing of the COL7A1 pre-mRNA.

A Splice Intervention Therapy for Autosomal Recessive Juvenile Parkinson's Disease Arising from Parkin Mutations.

Parkin-type autosomal recessive juvenile-onset Parkinson's disease is caused by mutations in the PRKN gene and accounts for 50% of all autosomal recessive Parkinsonism cases. Parkin is a neuroprotective protein that has dual functions as an E3 ligase in the ubiquitin-proteasome system and as a transcriptional repressor of p53. While genomic deletions of PRKN exon 3 disrupt the mRNA reading frame and result in the loss of functional parkin protein, deletions of both exon 3 and 4 maintain the reading frame and are associated with a later onset, milder disease progression, indicating this particular isoform retains some function. Here, we describe in vitro evaluation of antisense oligomers that restore functional parkin expression in cells derived from a Parkinson's patient carrying a heterozygous PRKN exon 3 deletion, by inducing exon 4 skipping to correct the reading frame. We show that the induced PRKN transcript is translated into a shorter but semi-functional parkin isoform able to be recruited to depolarised mitochondria, and also transcriptionally represses p53 expression. These results support the potential use of antisense oligomers as a disease-modifying treatment for selected pathogenic PRKN mutations.

Removal of the Polyglutamine Repeat of Ataxin-3 by Redirecting pre-mRNA Processing.

Spinocerebellar ataxia type 3 (SCA3) is a devastating neurodegenerative disease for which there is currently no cure, nor effective treatment strategy. One of nine polyglutamine disorders known to date, SCA3 is clinically heterogeneous and the main feature is progressive ataxia, which in turn affects speech, balance and gait of the affected individual. SCA3 is caused by an expanded polyglutamine tract in the ataxin-3 protein, resulting in conformational changes that lead to toxic gain of function. The expanded glutamine tract is located at the 5' end of the penultimate exon (exon 10) of ATXN3 gene transcript. Other studies reported removal of the expanded glutamine tract using splice switching antisense oligonucleotides. Here, we describe improved efficiency in the removal of the toxic polyglutamine tract of ataxin-3 in vitro using phosphorodiamidate morpholino oligomers, when compared to antisense oligonucleotides composed of 2'-O-methyl modified bases on a phosphorothioate backbone. Significant downregulation of both the expanded and non-expanded protein was induced by the morpholino antisense oligomer, with a greater proportion of ataxin-3 protein missing the polyglutamine tract. With growing concerns over toxicity associated with long-term administration of phosphorothioate oligonucleotides, the use of a phosphorodiamidate morpholino oligomer may be preferable for clinical application. These results suggest that morpholino oligomers may provide greater therapeutic benefit for the treatment of spinocerebellar ataxia type 3, without toxic effects.

Systematic Approach to Developing Splice Modulating Antisense Oligonucleotides.

The process of pre-mRNA splicing is a common and fundamental step in the expression of most human genes. Alternative splicing, whereby different splice motifs and sites are recognised in a developmental and/or tissue-specific manner, contributes to genetic plasticity and diversity of gene expression. Redirecting pre-mRNA processing of various genes has now been validated as a viable clinical therapeutic strategy, providing treatments for Duchenne muscular dystrophy (inducing specific exon skipping) and spinal muscular atrophy (promoting exon retention). We have designed and evaluated over 5000 different antisense oligonucleotides to alter splicing of a variety of pre-mRNAs, from the longest known human pre-mRNA to shorter, exon-dense primary gene transcripts. Here, we present our guidelines for designing, evaluating and optimising splice switching antisense oligomers in vitro. These systematic approaches assess several critical factors such as the selection of target splicing motifs, choice of cells, various delivery reagents and crucial aspects of validating assays for the screening of antisense oligonucleotides composed of 2'-O-methyl modified bases on a phosphorothioate backbone.

In Vitro Validation of Phosphorodiamidate Morpholino Oligomers.

One of the crucial aspects of screening antisense oligonucleotides destined for therapeutic application is confidence that the antisense oligomer is delivered efficiently into cultured cells. Efficient delivery is particularly vital for antisense phosphorodiamidate morpholino oligomers, which have a neutral backbone, and are known to show poor gymnotic uptake. Here, we report several methods to deliver these oligomers into cultured cells. Although 4D-Nucleofector™ or Neon™ electroporation systems provide efficient delivery and use lower amounts of phosphorodiamidate morpholino oligomer, both systems are costly. We show that some readily available transfection reagents can be used to deliver phosphorodiamidate morpholino oligomers as efficiently as the electroporation systems. Among the transfection reagents tested, we recommend Lipofectamine 3000™ for delivering phosphorodiamidate morpholino oligomers into fibroblasts and Lipofectamine 3000™ or Lipofectamine 2000™ for myoblasts/myotubes. We also provide optimal programs for nucleofection into various cell lines using the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza), as well as antisense oligomers that redirect expression of ubiquitously expressed genes that may be used as positive treatments for human and murine cell transfections.

Design of a framework for the deployment of collaborative independent rare disease-centric registries: Gaucher disease registry model.

Orphan drug clinical trials often are adversely affected by a lack of high quality treatment efficacy data that can be reliably compared across large patient cohorts derived from multiple governmental and country jurisdictions. It is critical that these patient data be captured with limited corporate involvement. For some time, there have been calls to develop collaborative, non-proprietary, patient-centric registries for post-market surveillance of aspects related to orphan drug efficacy. There is an urgent need for the development and sustainable deployment of these 'independent' registries that can capture comprehensive clinical, genetic and therapeutic information on patients with rare diseases. We therefore extended an open-source registry platform, the Rare Disease Registry Framework (RDRF) to establish an Independent Rare Disease Registry (IRDR). We engaged with an established rare disease community for Gaucher disease to determine system requirements, methods of data capture, consent, and reporting. A non-proprietary IRDR model is presented that can serve as autonomous data repository, but more importantly ensures that the relevant data can be made available to appropriate stakeholders in a secure, timely and efficient manner to improve clinical decision-making and the lives of those with a rare disease.

Primary over-expression of AßPP in muscle does not lead to the development of inclusion body myositis in a new lineage of the MCK-AßPP transgenic mouse.

The aim of this study is to determine whether primary over-expression of AßPP in skeletal muscle results in the development of features of inclusion body myositis (IBM) in a new lineage of the MCK-AßPP transgenic mouse. Quantitative histological, immunohistochemical and western blotting studies were performed on muscles from 3 to 18 month old transgenic and wild-type C57BL6/SJL mice. Electron microscopy was also performed on muscle sections from selected animals. Although western blotting confirmed that there was over-expression of full length AßPP in transgenic mouse muscles, deposition of amyloid-ß and fibrillar amyloid could not be demonstrated histochemically or with electron microscopy. Additionally, other changes typical of IBM such as rimmed vacuoles, cytochrome C oxidase-deficient fibres, upregulation of MHC antigens, lymphocytic inflammatory infiltration and T cell fibre invasion were absent. The most prominent finding in both transgenic and wild-type animals was the presence of tubular aggregates which was age-related and largely restricted to male animals. Expression of full length AßPP in this MCK-AßPP mouse lineage did not reach the levels required for immunodetection or deposition of amyloid-ß as in the original transgenic strains, and was not associated with the development of pathological features of IBM. These negative results emphasise the potential pitfalls of re-deriving transgenic mouse strains in different laboratories.

Regulation of eukaryotic gene expression by the untranslated gene regions and other non-coding elements.

There is now compelling evidence that the complexity of higher organisms correlates with the relative amount of non-coding RNA rather than the number of protein-coding genes. Previously dismissed as "junk DNA", it is the non-coding regions of the genome that are responsible for regulation, facilitating complex temporal and spatial gene expression through the combinatorial effect of numerous mechanisms and interactions working together to fine-tune gene expression. The major regions involved in regulation of a particular gene are the 5' and 3' untranslated regions and introns. In addition, pervasive transcription of complex genomes produces a variety of non-coding transcripts that interact with these regions and contribute to regulation. This review discusses recent insights into the regulatory roles of the untranslated gene regions and non-coding RNAs in the control of complex gene expression, as well as the implications of this in terms of organism complexity and evolution.

DNAzymes: Expanding the Potential of Nucleic Acid Therapeutics.

Nucleic acids drugs have been proven in the clinic as a powerful modality to treat inherited and acquired diseases. However, key challenges including drug stability, renal clearance, cellular uptake, and movement across biological barriers (foremost the blood-brain barrier) limit the translation and clinical efficacy of nucleic acid-based therapies, both systemically and in the central nervous system. In this study we provide an overview of an emerging class of nucleic acid therapeutic, called DNAzymes. In particular, we review the use of chemical modifications and carrier molecules for the stabilization and/or delivery of DNAzymes in cell and animal models. Although this review focuses on DNAzymes, the strategies described are broadly applicable to most nucleic acid technologies. This review should serve as a general guide for selecting chemical modifications to improve the therapeutic performance of DNAzymes.

A spotter's guide to SNPtic exons: The common splice variants underlying some SNP-phenotype correlations.

BACKGROUND: Cryptic exons are typically characterised as deleterious splicing aberrations caused by deep intronic mutations. However, low-level splicing of cryptic exons is sometimes observed in the absence of any pathogenic mutation. Five recent reports have described how low-level splicing of cryptic exons can be modulated by common single-nucleotide polymorphisms (SNPs), resulting in phenotypic differences amongst different genotypes. METHODS: We sought to investigate whether additional 'SNPtic' exons may exist, and whether these could provide an explanatory mechanism for some of the genotype-phenotype correlations revealed by genome-wide association studies. We thoroughly searched the literature for reported cryptic exons, cross-referenced their genomic coordinates against the dbSNP database of common SNPs, then screened out SNPs with no reported phenotype associations. RESULTS: This method discovered five probable SNPtic exons in the genes APC, FGB, GHRL, MYPBC3 and OTC. For four of these five exons, we observed that the phenotype associated with the SNP was compatible with the predicted splicing effect of the nucleotide change, whilst the fifth (in GHRL) likely had a more complex splice-switching effect. CONCLUSION: Application of our search methods could augment the knowledge value of future cryptic exon reports and aid in generating better hypotheses for genome-wide association studies.

Corrigendum: Analysis of Pathogenic Pseudoexons Reveals Novel Mechanisms Driving Cryptic Splicing.

[This corrects the article DOI: 10.3389/fgene.2021.806946.].

Primary Nasal Epithelial Cells as a Surrogate Cell Culture Model for Type-II Alveolar Cells to Study ABCA-3 Deficiency.

ATP Binding Cassette Subfamily A Member 3 (ABCA-3) is a lipid transporter protein highly expressed in type-II alveolar (AT-II) cells. Mutations in ABCA3 can result in severe respiratory disease in infants and children. To study ABCA-3 deficiency in vitro, primary AT-II cells would be the cell culture of choice although sample accessibility is limited. Our aim was to investigate the suitability of primary nasal epithelial cells, as a surrogate culture model for AT-II cells, to study ABCA-3 deficiency. Expression of ABCA3, and surfactant protein genes, SFTPB and SFTPC, was detected in primary nasal epithelial cells but at a significantly lower level than in AT-II cells. ABCA-3, SP-B, and SP-C were detected by immunofluorescence microscopy in primary nasal epithelial cells. However, SP-B and SP-C were undetectable in primary nasal epithelial cells using western blotting. Structurally imperfect lamellar bodies were observed in primary nasal epithelial cells using transmission electron microscopy. Functional assessment of the ABCA-3 protein demonstrated that higher concentrations of doxorubicin reduced cell viability in ABCA-3 deficient nasal epithelial cells compared to controls in an assay-dependent manner. Our results indicate that there may be a role for primary nasal epithelial cell cultures to model ABCA-3 deficiency in vitro, although additional cell culture models that more effectively recapitulate the AT-II phenotype may be required.