Towards personalized therapies for genetic disorders of surfactant dysfunction

Abstract

Genetic disorders of surfactant dysfunction are a rare cause of chronic, progressive or refractory respiratory failure in term and preterm infants. This review explores genetic mechanisms underpinning surfactant dysfunction, highlighting specific surfactant-associated genes including SFTPB, SFTPC, ABCA3, and NKX2.1. Pathogenic variants in these genes contribute to a range of clinical presentations and courses, from neonatal hypoxemic respiratory failure to childhood interstitial lung disease and even adult-onset pulmonary fibrosis. This review emphasizes the importance of early recognition, thorough phenotype assessment, and assessment of variant functionality as essential prerequisites for treatments including lung transplantation. We explore emerging treatment options, including personalized pharmacological approaches and gene therapy strategies. In conclusion, this comprehensive review offers valuable insights into the pathogenic mechanisms of genetic disorders of surfactant dysfunction, genetic fundamentals, available and emerging therapeutic options, and underscores the need for further research to develop personalized therapies for affected infants and children.

Introduction

Respiratory distress syndrome (RDS) is the consequence of surfactant insufficiency, resulting in increased alveolar surface tension and airspace collapse. In premature infants, RDS primarily arises due to alveolar epithelium immaturity and insufficient surfactant production¹. However, regardless of gestational age, chronic, progressive, or refractory RDS may result from genetic diseases of surfactant dysfunction^2–4. Pathogenic variants in several genes, pivotal for maintaining surfactant homeostasis and function, have been identified as rare yet significant contributors to a wide spectrum of chronic lung diseases, ranging from lethal hypoxemic respiratory failure (HRF) in term and preterm neonates to childhood interstitial lung disease (chILD) and even adult-onset pulmonary fibrosis. Despite the current lack of effective therapeutic options, several pre-clinical studies are delineating potential strategies for personalized pharmacologic or genetic treatments. Early recognition of the phenotype, identification of the molecular etiology, and variant functional characterization are important steps to facilitate targeted interventions.

In this review, we aim to provide clinicians with an overview of the current knowledge on disease mechanisms, state-of-the-art management, and future therapies targeting molecular defects.

Surfactant structure, function, and metabolism

Pulmonary surfactant, composed of phospholipids (80%), neutral lipids (10%), and proteins (10%)⁵, lowers surface tension in the lung preventing atelectasis at end-expiration⁶, and acts as an innate immunity mechanism⁷. Pulmonary surfactant is assembled and stored within the lamellar bodies of alveolar type 2 pneumocytes (AEC2)⁸. Surfactant is secreted from the apical pole as tubular myelin⁹, then spreads at the air-liquid interface of the alveolar epithelium, forming a dynamic monolayer expanding and contracting over repeated breathing cycles, optimizing surface tension to prevent alveolar collapse and/or overdistension⁸. Intra-alveolar surfactant homeostasis is maintained by net contributions from synthesis, secretion, uptake, and catabolism by AEC2s and alveolar macrophages^{5, 10}. Figure 1 summarizes the surfactant pathway from gene expression to surfactant assembly, secretion, and degradation.

Figure 1:

Genetic disorders of surfactant dysfunction: current and emerging treatments

Surfactant proteins B and C (SP-B, SP-C), encoded by the genes SFTPB and SFTPC, are extremely hydrophobic proteins and contribute to the surface tension lowering qualities of surfactant. SP-B is critical for surfactant packaging, adsorption, and surface tension-lowering properties^{11, 12}. SP-C plays a less critical role at birth, contributing to surface film formation and stability, especially at alveolar near-collapse^{13, 14}.

In addition to surfactant proteins, surfactant lipids are critical for surface activity and stability¹⁵. ATP-Binding cassette transporter A3 (ABCA3) is a lamellar body transmembrane phospholipid transporter essential for organelle biogenesis, surfactant protein-phospholipid assembly, and alveolar homeostasis¹⁶. Thyroid transcription factor 1 (TTF–1), encoded by NKX2.1, plays a central role in lung morphogenesis, epithelial differentiation, and up-regulation of most surfactant genes (SFTPA, SFTPB, SFTPC, SFTPD, ABCA3), in addition to development of the thyroid gland and central nervous system. Finally, surfactant clearance by alveolar macrophages is also a critical step in alveolar homeostasis, for which Granulocyte macrophage-colony stimulating factor (GM-CSF) signaling plays a central role¹⁷.

Genetic diseases of the surfactant system

1). Surfactant protein B deficiency

SP-B deficiency is an autosomal recessive disease and occurs ~1 per million births¹⁸. Infants typically present with neonatal HRF and die within the first months of life without lung transplantation^{4, 19}. Surfactant replacement has only transient effects on oxygenation and ventilation and does not result in prolonged survival²⁰. Most affected infants have recessively inherited biallelic null variants of SFTPB leading to complete or near complete loss of mature SP-B expression^{4, 21}. The frameshift variant p.Pro133Glnfs*95 [c.361delinsGAA;“121ins2”] is identified in over 70% of affected infants²¹. This variant introduces a premature stop codon resulting in undetectable levels of SP-B in lung tissue and alveolar fluid, as well as proSP-C misprocessing²². In rare instances, prolonged survival beyond the first months or years has been associated with hypomorphic missense variants resulting in some residual SP-B expression and function^{23, 24}.

2). Surfactant protein C-associated lung disease

SFTPC pathogenic variants are associated with a wide spectrum of clinical presentations, ranging from lethal hypoxemic respiratory failure in neonates²⁵ to, most typically, chILD and adult-onset pulmonary fibrosis^{26, 27}. SP-C -associated lung disease is caused by monoallelic usually missense variants, about half of which are dominantly inherited with variable penetrance, and half arise de novo. Surfactant dysfunction results from dominant-negative or toxic gain-of-function mechanisms. The distal C-terminus sequence of the apoprotein contains a crucial BRICHOS domain which functions as a chaperone to prevent aggregation of the highly hydrophobic mature peptide during post-transcriptional processes. BRICHOS variants lead to misfolding, activation of the unfolded protein response, and cell death^{25, 28}. Pathogenic variants in the apoprotein linker sequence, including the most frequent p.Ile73Thr, cause abnormal intracytoplasmic trafficking and accumulation of unprocessed pro-peptide fragments in the plasma membrane and endosomes^{25, 29}. Missense variants affecting the mature peptide domain, as well as splicing variants and large copy number variants are only rarely reported³⁰.

3). ATP-Binding cassette A3 deficiency

ABCA3 deficiency is the most frequent among the genetic diseases of surfactant dysfunction, typically presenting as neonatal HRF similar to SP-B deficiency, or less commonly as chILD frequently evolving toward end-stage lung disease in the first months or years of life^31–33. ABCA3 deficiency is an autosomal recessive disease and results from biallelic variants of ABCA3. Over 300 disease-associated variants including nonsense, frameshift, missense, splice site, and small insertion/deletion (‘indel’) variants have been reported to date^34–36. ABCA3 missense variants may affect ABCA3 trafficking to the lamellar body membrane (class I) or may disrupt ATP hydrolysis and lipid transport (class II). As most affected patients are compound heterozygous, and ABCA3 variants are often private, disease severity is difficult to predict and will depend on the combined residual function resulting from the two variant alleles^{34, 35}.

Single ABCA3 variants, may result in reversible RDS among term and late preterm infants^37–39. Some infants have been identified to carry only a single ABCA3 variant, but express the full ABCA3 deficiency phenotype, suggesting non-coding variants of the opposite allele, which may not be detected with exon-based sequencing, may alter gene expression. Uniparental disomy has been occasionally described as a potential disease mechanism⁴⁰, with a low risk of recurrence in future pregnancies. The recurrent deep intronic variant IVS25–98T, resulting in aberrant splicing, was identified in several affected infants in whom only one heterozygous coding variant had been found⁴¹. Large intragenic deletions spanning entire exons undetectable by Sanger sequencing have also been reported³⁶. In addition, heterozygous variants in ABCA3 may modify disease presentation or severity among individuals with SFTPC variants⁴².

4). Thyroid transcription factor 1 associated lung disease

TTF-1 is a small protein expressed in the lung, thyroid, basal ganglia, and hypothalamus, and plays multiple roles in organ formation and function⁴³. TTF-1 -associated lung disease combines features of surfactant dysfunction and impaired alveolarization⁴⁴. Monoallelic NKX2.1 variants may be associated with the brain-lung-thyroid syndrome, a complex phenotype that includes various combinations and degrees of hypothyroidism, benign hereditary chorea, acinar dysplasia, neonatal HRF, and chILD^{45, 46}. Single nucleotide, small indels and large copy number variants, dominantly inherited or arising de novo, have been reported. Penetrance varies widely, and individuals with the same variant may display varying degrees of neurologic abnormalities, thyroid disease, and/or pulmonary disease, thus making it challenging to predict their individual clinical progression.

5). Other surfactant-related genetic diseases

Rare variants in additional genes have been described in infants with clinical phenotypes of surfactant dysfunction. Variants of the GM-CSF receptors subunits A (CSF2RA)⁴⁷ and B (CSF2RB)^{48, 49} manifest as pulmonary alveolar proteinosis (PAP), albeit presenting in late infancy or childhood. Methionyl-tRNA synthetase-1 (MARS1) variants are another ultrarare cause of early-onset PAP associated with liver disease, most frequently identified among individuals from Reunion island⁵⁰. A RAB5B variant disrupting SP-B and -C intracytoplasmic processing was recently identified in an infant with fatal ILD, neurodevelopmental delay, and dysmorphic features⁵¹. Filamin A (FLNA) variants cause a severe form of chILD that may present in the neonatal period, typically associated with pulmonary hypertension, dysmorphic features, and periventricular nodular heterotopia⁵². Variants in SFTPA1 and SFTPA2 are associated with adult-onset ILD and lung cancer⁵³. These diseases have distinctive features, but their prevalence and potential role in infant and childhood surfactant dysfunction disorders have not been thoroughly explored.

Figure 2 shows radiographic phenotypes of several genetic diseases of surfactant dysfunction.

Figure 2. Clinical evolution of surfactant dysfunction disease.

A. Chest radiograph in a term infant on day 14 of life with ABCA3 deficiency showing mild diffuse hazy granular airspace opacification resembling the findings of respiratory distress syndrome of prematurity. – B and C. Same infant’s chest radiograph at 2 (B) and 4 (C) months, showing progressive airspace coarsening and septal thickening. – D. Chest CT of a 4-month-old infant with ABCA3 deficiency showing ground-glass opacity, patchy cystic areas, and peribronchial and interlobar thickening. – E. Chest CT of a 6-month-old infant with an SFTPC variant with mild chronic lung disease showing mild interstitial thickening and small subpleural and lobular cysts in clusters. – F. Chest CT of an infant with NKX2.1 variant at 1 month of life, showing diffuse ground glass attenuation and mild septal thickening.

Treatment approaches for genetic disorder of surfactant dysfunction

Table 1 summarizes the genetic mechanisms and histopathology phenotypes associated with pathogenic variants in SFTPB, SFTPC, ABCA3, and NKX2.1 as well as potential treatment options. Infants with SP-B or ABCA3 deficiency due to biallelic loss-of-function variants present with neonatal respiratory failure that is usually progressive despite pharmacological interventions^{19, 21, 34, 35}. The clinical courses of infants and children with pathogenic variants in SFTPC and NKX2–1 as well as missense variants in ABCA3 are more variable and difficult to predict. Lower respiratory tract infections and ILD exacerbations may trigger long-term deteriorations of the clinical course⁵⁴.

Table 1.

Summary of surfactant deficiencies

	Surfactant protein B	Surfactant protein C	ATP-binding cassette A3	Thyroid transcription factor 1
Estimated incidence	1/1,000,000	Unknown	1/90,000 births	Unknown
Gene	SFTPB	SFTPC	ABCA3	NKX2.1
Genomic position	2p11.2	8p21.3	16p13.3	14q13
Heritability	Autosomal recessive with complete penetrance Mostly inherited	Autosomal dominant with variable penetrance (~50%) Inherited or de novo (~50%)	Autosomal recessive Mostly inherited	• Autosomal dominant with variable penetrance • Inherited or de novo
Molecular genetics	>40 SNVs: most frequently null variant p.Pro133Glnfs*95 (“121ins2”) (~70%)	>60 SNVs: LINKER propeptide domain; most frequent variant: p.Ile73Thr (25%) BRICHOS propeptide domain Rarely, mature peptide Rare large CNV	>300 SNVs, mostly private: Null variants Most common pathogenic variant: p.Glu292Val Rare large CNV	>80 variants: SNVs: null and hypomorphic variants mostly in the DNA-binding homeodomain CNV (~15%)
Disease mechanisms	Loss of SP-B expression	Disrupted intracellular trafficking (LINKER variants) Protein misfolding, endoplasmic reticulum stress (BRICHOS variants)	Loss of ABCA3 expression Disrupted phospholipid transport Disrupted intracellular trafficking	Decreased expression of surfactant-related genes (SFTPA, SFTPB, SFTPC, ABCA3) Altered lung development (branching and differentiation)
Clinical presentations	Neonatal hypoxemic respiratory failure (most) Childhood chronic lung disease (very rare)	Neonatal hypoxemic respiratory failure (~10%) Infantile (~40%) and childhood ILD Recurrent respiratory infections Adult idiopathic pulmonary fibrosis	Neonatal hypoxemic respiratory failure Infantile ILD	Neonatal hypoxemic respiratory failure Childhood ILD Hypothyroidism Benign familial chorea, hypotonia Lung, thyroid, and blood cancers
Current therapies	Early transplant, neonatal or infancy	Hydroxychloroquine Azithromycin Steroids Infant/childhood transplant	Steroids Hydroxychloroquine Azithromycin Infant/childhood transplant	Steroids Hydroxychloroquine Azithromycin

1). Nonspecific pharmacological approaches

Medical therapies for genetic diseases of surfactant dysfunction remain limited and nonspecific^{35, 55}. Affected infants and children primarily receive supportive care, including oxygen supplementation, chronic ventilation, and nutritional supplementation. Current pharmacologic treatments are used without distinction based on the underlying molecular mechanism and include systemic glucocorticoids, hydroxychloroquine, and azithromycin. Exogenous surfactant has been used in infants with neonatal HRF and may bring some transient improvement in oxygenation and ventilation. These empiric treatments can be given alone or in combination, and response varies with the patient’s age, the presence of inflammatory or fibrotic lung disease, and the molecular diagnosis⁵⁵. Monitoring treatment response is crucial and involves iterative assessment of respiratory function, activity, growth, and development at baseline and during follow up⁵⁶.

In a recent European expert opinion group Delphi review, the consensus was that infants with chILD who are ventilated or at risk of requiring ventilation show a response to high-dose glucocorticoid “pulse therapy” (10 mg/kg methylprednisolone once daily for 3 days) within 12 to 48 hours, reaching full effect within a week⁵⁷. In contrast, hydroxychloroquine may take 2–4 weeks to demonstrate noticeable effects. Non-ventilated children typically may show slower clinical responses, requiring up to 4 weeks for glucocorticoids, 12 weeks for hydroxychloroquine or azithromycin in order to assess treatment effects. However, the available literature is hampered by limited data and significant publication bias, especially regarding azithromycin. Clinical practices and protocols rely heavily on small case series and expert opinions, calling for a nuanced interpretation of findings^{58, 59}. Within these limitations, studies have suggested some efficacy in subsets of patients with SP-C-related disease⁵⁵ and, more limitedly, with ABCA3 deficiency⁶⁰. Recently, a first double-blinded, randomized, placebo-controlled trial of hydroxychloroquine in chILD that included, among others, 15 children with a genetic disease of surfactant dysfunction did not show significant oxygen level improvement with treatment⁵⁹. However, this study was limited by a relatively small sample size and lacked lung function testing in children under 6 years old, warranting caution in interpreting the potential benefits of hydroxychloroquine in this group. Altogether, these treatments, albeit based on anecdotal evidence, are applied empirically, alone or combined, in seriously or chronically sick children by various clinicians, and remain the mainstay of current pharmacological therapy for chILD.

2). Prevention of lung fibrosis

Surviving children and adolescents with genetic surfactant dysfunction diseases may develop pulmonary fibrosis and chronic, progressive respiratory failure. While pirfenidone and nintedanib are approved for adults with fibrosing interstitial lung disease (ILD), their use in children is not established⁶¹. A recent randomized trial of nintedanib for 24 weeks in 39 patients (age 6–17 years) with fibrosing chILD, including 12 patients with genetic surfactant dysfunction diseases, yielded favorable safety results, but efficacy assessment was not the primary focus⁶¹.

3). Lung transplant

Lung transplantation has been performed since the 1990s for progressive respiratory failure among infants and children with genetic surfactant dysfunction diseases. Evaluation for lung transplantation relies strongly on genetic testing, and advances in next-generation sequencing (gene panels, exome, or whole genome), allowing fast precision diagnosis, has reduced the need for lung biopsy⁶². Genetic results must be interpreted carefully in collaboration with pediatric pulmonologists and clinical geneticists, as some genetic surfactant dysfunction diseases may not necessarily be lethal without lung transplant (i.e., pathogenic variants in SFTPC and NKX2-1, or missense variants in ABCA3). Parental testing may be required in order to determine whether SFTPB or ABCA3 variants are inherited in trans (both alleles affected) or in cis (single allele affected)³⁴.

Pathogenic or likely pathogenic variants may be identified based on the predicted effects on protein function (e.g., nonsense, frameshift variants) or previous identification in unrelated symptomatic individuals^{34, 60}. For conditions like ABCA3 deficiency, the majority of disease-associated variants are missense variants, often unique to an affected individual or family (“private”), thus are often categorized as variants of uncertain significance (VUS)^{34, 60}. Interpretation of VUS remains a significant challenge to the field of medical genetics. When genetic testing results are inconclusive or incongruent with the patient’s clinical course, specialists may pursue lung biopsy, which carries risk for morbidities including air leak and bleeding.

Parental education is crucial when considering lung transplantation for infants and children, as it comes with significant early and late complications. Despite advances in diagnosis and treatment, the 5-year survival rate for lung transplants remains approximately 60% for infants and 80% for children^{19, 63}. Immediate post-transplant complications may include bleeding, anastomosis, or vascular issues, early graft dysfunction, and infections. Within the first 3 months, complications can involve rejection (T-cell or antibody-mediated) and viral or bacterial infections. Late complications, beyond 3 months, include chronic lung allograft dysfunction (bronchiolitis obliterans syndrome and restrictive allograft syndrome)⁶⁴, along with hypertension, renal insufficiency, post-transplant lymphoproliferative disorder, and diabetes¹⁹. Speech and motor developmental delays and hearing loss are especially common among those transplanted during infancy, likely reflecting the severity of disease at time of transplant¹⁹. Outcomes for infants and children with genetic surfactant dysfunction diseases are comparable to those who receive lung transplants for other indications⁶⁵. Nevertheless, due to the chronic, complex medical and surgical burden of pediatric lung transplantation, approximately half of families ultimately pursue comfort care over transplantation⁶⁶.

4). Targeted pharmacological therapies

Efforts are underway to develop variant-specific small molecules to treat genetic diseases of surfactant dysfunction, exemplified by the success in cystic fibrosis^{67, 68}. These efforts involve several steps: expressing the gene of interest with specific variants in a cellular model⁴, establishing relevant functional assessments for these variants, screening libraries of small molecules to correct their functional consequences⁴, optimizing compounds for safety and efficacy, and conducting in vivo studies and clinical trials to assess drug toxicity, pharmacokinetics, pharmacodynamics, and efficacy.

Hydroxychloroquine, empirically used in chILD, recently showed a efficacy for some ABCA3 variants⁶⁹. In vitro results showed varying degrees of improvement in ABCA3 proteolytic cleavage, ABCA3-positive vesicle volume, and phospholipid transport for different ABCA3 mutants (E292V, D953H, Q1045R, A1046E), suggesting variant-specific effects⁶⁹. This variant-specific hydroxychloroquine response demonstrated in vitro was in retrospect corroborated with clinical responses and outcomes in patients carrying these same variants.

ABCA3 shares common structural and functional features with other ABC family transporters, in particular ABCC7, also known as cystic fibrosis transmembrane conductance regulator (CFTR), for which pharmacological therapies specific to the underlying molecular defects have been extensively studied and are now approved for the treatment of patients with cystic fibrosis^{67, 68}. Compounds enhancing channel function are designated as potentiators. Chemical or pharmacological chaperones restoring folding and trafficking, thereby increasing the amount of protein available at the plasma membrane, are designated as correctors. Several ABCA3 Class II (phospholipid transport) mutants transfected in a cell line model were functionally rescued with the CFTR potentiators ivacaftor and genistein⁷⁰. Bithiazole correctors C13 and C17, and the chemical chaperone trimethylamine N-oxide showed promise in correcting some class I (trafficking) mutants in vitro⁷¹. These results point towards the possibility that clinically approved CFTR correctors and potentiators may be repurposed for some individuals with ABCA3 deficiency, depending on genotype.

A high-throughput screening platform identified Cyclosporine A as a potential corrector for specific ABCA3 mutants⁷². While these studies showed improved proteolytic processing and phosphatidylcholine uptake into ABCA3 positive vesicles, they did not assess actual secretion, composition, and biophysical activity of the surfactant.

In desperate clinical situations, these repurposed drugs with known safety profiles like hydroxychloroquine, cyclosporine, or the CFTR modulators may be considered in an attempt to improve clinical course. However, prospective randomized trials are most desirable and need to be implemented with innovative designs.

5). Gene therapy

There have been major advances in gene addition and genome editing strategies that have been applied to human cell lines and model organisms to characterize and even rescue surfactant deficiency or dysfunction. While these discoveries hold tremendous promise for treatment of genetic surfactant diseases, the approaches will likely be gene-specific due to differences in pathogenic mechanisms, gene size, and associated phenotypes.

These advances have brought hopes of correcting gene variations by replacing or repairing the gene to correct cellular and organ function. Extensive research^73–76 is focused on developing delivery systems (“vectors”) for DNA, antisense oligonucleotides, peptides, long noncoding RNA (lncRNA), or small interfering RNA (siRNA) to target specific organs and cell types involved in disease. Adenoviruses (AV) and adeno-associated viruses (AAV), are widely studied viral vectors. AV is versatile, efficient, and can carry large transgenes but may trigger inflammation and immune clearance, while AAV is less inflammatory but has limitations in gene size and remains transient due to lack of transfection efficiency. Several lentiviruses and retroviruses can insert DNA into human chromosomes, providing a long-term correction strategy. In addition to viral vectors, nanoparticles can also deliver therapeutic cargoes, improving targeting and transfection, though less efficiently and durably than viral vectors¹⁸.

In loss-of-function diseases like SP-B and ABCA3 deficiencies, gene therapy aims to restore native gene expression efficiently and persistently. Gene addition, involving the introduction of a full-length functional coding sequence, is a potential strategy. SFTPB has a relatively short coding sequence which makes it amenable to several types of vectors, including AAV which has been approved for clinical use for other indications⁷³. Intratracheal administration of a lung-tropic AAV vector delivering murine or human SFTPB cDNA into SP-B deficient mice restored surfactant homeostasis, prevented lung injury, and improved lung physiology and survival⁷⁷. A lentiviral vector expressing SP-B was used to rescue an in vitro model of SP-B deficiency in human alveolar epithelial cells⁷⁸. In a human lung organoid model derived from patient-specific induced pluripotent stem cells, reversal of the SP-B deficiency phenotype was obtained by gene addition using a lentivirus carrying the wild type SFTPB cDNA⁷⁹.

For ABCA3, the longer coding sequence may require the use of different vectors allowing larger cargoes. Lentiviral vectors like rSIV.F/HN showed promise in CFTR gene therapy⁸⁰ and could be applied in the future for ABCA3 gene therapy.

SP-C-associated ILD, a dominantly inherited gain-of-function disease, requires a different approach. Gene knockout/knockdown, RNA interference, and antisense RNA can decrease or silence mutated SFTPC peptide expression. CRISPR/Cas9 nuclease-mediated gene knockout has shown success in mice⁸¹. AAV-packaged CRISPR/Cas9-based tools may repair individual missense variants¹⁸ offering a personalized approach for surfactant-related gene variants, but require precise diagnosis and functional studies.

Tools to study genetic diseases of surfactant dysfunction

Translational research needs to model various aspects of surfactant-related physiology, pathology and therapy in order to develop personalized therapies in human. Mutagenesis techniques provide rapid and reproducible assays for functional testing of individual variants in commercially available cell lines. Induced pluripotent stem cells from affected infants allow for more accurate modeling of individual disease without the need for genetic manipulation. Finally, animal models represent the ultimate preclinical step for testing targeted therapies.

1). Cell transfection and transduction for in vitro modelling

Human cell lines, including A549, H441, and HEK293 cells derived from pulmonary adenocarcinoma and embryonic kidney, have been used to model genetic diseases of surfactant dysfunction in vitro through techniques such as transfection (stable or transient) or viral transduction.^{39, 82–85}. These cell models offer valuable insights into disease mechanisms of surfactant-associated genes and variants, although do not fully recapitulate all AEC2 properties. Notably, they provide efficient, easy-to-maintain, and readily available platforms for studying disease-associated variants in SFTPC and ABCA3. For example, assay systems have been developed to functionally characterize ABCA3 variants, including the expression, co-localization with intracellular organelles, phospholipid uptake, and ABCA3-positive vesicular size and ultrastructure and to assess in vitro responses to pharmacologic therapies^{69, 71, 72, 82, 86–89}.

2). Induced Pluripotent Stem Cells and Genome Editing

In addition to immortalized cell lines, induced pluripotent stem cells (iPSCs) derived from affected infants and children with genetic diseases of surfactant dysfunction have been differentiated into AEC2-like cells in vitro^{90, 91}. This approach allows investigators to model disease mechanisms and test potential therapies. For example, iPSCs from individuals with SP-B deficiency or SFTPC variants have been used to create iAEC2s with characteristics closely resembling disease phenotypes^{90, 91}. CRISPR/Cas9 genome editing has also been employed to correct these variants, restoring AEC2 cell function in vitro⁹⁰. Furthermore, these models have been used to assess the cellular impact in vitro of medications like hydroxychloroquine⁹¹.

3). Murine Models

Conditional deletions of SFTPB and ABCA3 in murine models have been used to investigate alveolar injury and regeneration as well as therapeutic interventions including AAV-based gene addition^{77, 92, 93}. Depending on the specific strain, SFTPC gene deletion in mice can lead to inflammation and increased disease susceptibility, particularly with viral respiratory infections⁹⁴. Recently, murine models expressing BRICHOS or non-BRICHOS SFTPC variants have been developed, resulting in spontaneous inflammation and lung fibrosis^{95, 96}. Mouse models with homozygous deletions of SFTPB and ABCA3 display lethal neonatal respiratory failure shortly after birth, closely resembling the human diseases^{92, 97, 98}. Mice homozygous for the most common pathogenic ABCA3 variant p.Glu292Val show age-dependent inflammation and altered macroautophagy in AEC2s, along with collagen deposition in the alveolar septa⁹⁹. In utero CRISPR/Cas9 genome editing to inactivate non-BRICHOS p.Ile73Thr mutant alleles improved lung morphology and survival in treated pups⁸¹. These mouse models play a crucial role in understanding disease mechanisms and evaluating potential therapies.

4). International registries

Finally, international registries and research networks, such as the European Research Collaboration for Children’s Interstitial Lung Disease (chILD-EU, http://www.childeu.net) and the U.S. National Registry for Childhood Interstitial and Diffuse Lung Disease registry¹⁰⁰ play important roles in advancing knowledge, promoting research, improving care, and providing support and resources to clinicians, patients, and families in the quest for therapies.

Conclusion

Genetic surfactant dysfunction diseases are rare, have provided important insights into normal human lung development and function, and are targets for promising therapeutic interventions. Rapid identification of the underlying molecular defect is essential for the diagnosis and is highly relevant for a personalized therapeutic approach. Nonspecific pharmacological therapies currently remain the mainstay for these disorders. Personalized therapies targeting specific variants hold promise, but innovative trial concepts are necessary due to the rarity of cases. Gene therapies tailored to address specific molecular defects may offer the perspective of a cure, but research is still at the preclinical stage. Patients affected with these complex diseases benefit from highly specialized centers offering specific expertise, advanced diagnostic resources in genetics, pathology, and imaging, and multidisciplinary care for acute and chronic management, including lung transplantation.

Practice points:

• Genetic disorders of surfactant dysfunction are a group of severe or lethal rare diseases with a clinical spectrum spanning from birth to adulthood

• Early recognition and precision diagnosis are essential for care orientation and treatment

• Personalized therapies offer new perspectives of treatment and potential cure but are still at the preclinical stage

Research directions:

• Improve and implement tools for functional characterization of surfactant gene variants

• Develop and implement vectors and technologies for safe human gene therapies

• Design and implement innovative clinical trials for targeted pharmacological and genetic therapies