Mechanisms and Targeted Therapy of Airway Basal Cell Dysfunction in Bronchiolitis Obliterans Syndrome

NCT07018804 | Enrolling By Invitation DMC

• 1 other identifier: SC20250160
• Study Type: interventional
• Target: 5
• Locations: 1 country
• Sites: 1

Brief Summary

This experimental study aims to investigate the pathogenesis of bronchiolitis obliterans syndrome (BOS) and provide a basis for clinical diagnosis and treatment. The core research question is: whether there is a causal relationship between stem cell dysfunction induced by the inflammatory microenvironment and airway injury repair during the pathological process of BOS? Researchers will collect alveolar lavage fluid specimens from participants and healthy individuals to isolate distal small airway stem cells for subsequent scientific research and comparative analysis, thereby revealing the pathological mechanisms of BOS, exploring precise intervention targets, and developing innovative therapeutic strategies to improve patient prognosis, long-term survival rates, and quality of life.

Conditions

Bronchiolitis Obliterans Syndrome (BOS)

Keywords

Bronchiolitis Obliterans Syndrome; Airway epithelium; Airway basal cells; Inflammatory response

Outcome Measures

Primary Outcomes (4)

• The self - renewal ability of airway basal cells

  By comparing the Ki67 expression levels of airway basal cells and the results of the CCK8 assay in different samples (such as patients with bronchiolitis obliterans syndrome and healthy volunteers), clarify the abnormal manifestations of the self - renewal function of airway basal cells in the disease state, and further explore the internal connection between it and the occurrence and development of bronchiolitis obliterans syndrome.

  From enrollment to 32 weeks

• Differentiation ability of airway basal cells

  Collect the differentiation samples of the air - liquid interface (ALI) in vitro differentiation culture for 21 days and the severe combined immunodeficiency (NSG) mice in vivo differentiation for 28 days respectively. With the help of real - time fluorescence quantitative polymerase chain reaction (Real - time PCR) and immunofluorescence techniques, conduct a detailed analysis of the cell types and their proportions in the differentiated structures. By detecting the expression of ciliated cell marker Ace - Tubulin, goblet cell marker MUC5AC, etc., accurately evaluate the ability of airway basal cells to differentiate into different cell types, providing a key basis for a deep understanding of the mechanism of airway epithelial injury repair.

  From enrollment to 32 weeks

• Survival rate of ferret experiments

  In the ferret airway basal cell transplantation experiment, closely monitor the survival rate of ferrets, which is a key indicator for evaluating the impact of airway basal cell transplantation on the disease progression of bronchiolitis obliterans syndrome.

  From enrollment to 32 weeks

• Weight changes in ferret experiments

  In the experiment of ferret airway basal cell transplantation, the weight changes of ferrets are regularly recorded. The weight changes can intuitively reflect the overall health of ferrets and assist in judging the effect of airway basal cell transplantation.

  From enrollment to 32 weeks

Secondary Outcomes (2)

• Molecular characteristics of cells

  From enrollment to 32 weeks

• Microenvironment change

  From enrollment to 32 weeks

Study Arms (8)

Patient group

EXPERIMENTAL

Epithelial mucosal tissues from the diseased lung regions and relatively healthy lung lobes of BOS patients following hematopoietic stem cell transplantation (HSCT) will be collected. Alveolar lavage fluid specimens will be obtained when available. The intervention consists of digesting the collected samples with tissue collagenase for the culture of airway basal cells (BCs).

Procedure: Bronchoscopic brushing

Healthy control group

EXPERIMENTAL

Healthy volunteers with no obvious airway abnormalities will be selected to collect samples from the same anatomical sites. No disease-related interventions will be performed, serving as a comparative control to highlight the abnormal conditions of basal cells (BCs) in the patient group.

Procedure: Bronchoscopic brushing

Single - cell clone group

EXPERIMENTAL

Genetic: Single - cell cloning

Cell Function Identification Group

EXPERIMENTAL

Genetic: Cell function identification

Local microenvironment change identification group

EXPERIMENTAL

Genetic: Identification of local microenvironmental changes

Gene Editing Group

EXPERIMENTAL

Genetic: Gene editing

Ferret airway basal cell transplantation group (prevention group)

EXPERIMENTAL

Procedure: Prevention of ferret airway basal cell transplantation

Ferret airway basal cell transplantation group (treatment group)

EXPERIMENTAL

Procedure: Transplantation treatment of ferret airway basal cells

Interventions

Bronchoscopic brushing PROCEDURE

The collected samples are digested with tissue collagenase for the culture of airway basal cells (BCs).

Healthy control group; Patient group

Single - cell cloning GENETIC

After primary BCs are expanded in the P1 passage, single-cell cloning libraries are established by planting them into 384-well cell culture plates in a single-cell per well format using a flow chamber cell sorter. Ten samples are selected from the expandable clones for the identification of differentiation and proliferation capabilities, while the remaining clones are cryopreserved.

Single - cell clone group

Cell function identification GENETIC

For single-cell samples of each patient, in vitro expansion culture is performed to observe the morphology of cells at each passage and calculate the clonogenic rate. Immunofluorescence technique is used to detect the expression of the cell proliferation marker Ki67, and the proliferation capacity is evaluated by combining with the CCK8 assay. Cells at passages P3-P5 are seeded on the permeable membrane of cell culture inserts at a density of \\(10\^6\\) cells/cm², and after 21 days of culture, the differentiated structures are collected. The expression of ciliated cell marker Ace-Tubulin and goblet cell marker MUC5AC is detected to assess the differentiation capacity. Meanwhile, cells are injected subcutaneously into NSG mice at \\(10\^6\\) cells/injection site for in vivo differentiation for 28 days, and the differentiated structures are collected for pathological analysis.

Cell Function Identification Group

Identification of local microenvironmental changes GENETIC

The air-liquid interface (ALI) differentiation culture medium is collected, and cell debris is removed by high-speed centrifugation. The supernatant is then collected to extract proteins. Target proteins are purified via immunoprecipitation or affinity chromatography, followed by desalting and concentration for mass spectrometry analysis to detect inflammatory cytokines and extracellular matrix (ECM). Real-time PCR and Western blot techniques are used to measure the expression levels of epithelial markers, mesenchymal markers, and ECM in cells across all groups, thereby evaluating changes in the local microenvironment around small airways.

Local microenvironment change identification group

Gene editing GENETIC

After plasmid construction, based on gene function, the CRISPR-Cas9-sgRNA (all-in-one) plasmid is transiently transfected into expanded single-cell strains via Nucleofection to knockout the target gene, or the CRISPR-dCas9 fusion-sgRNA plasmid is transiently transfected to activate or inhibit the target gene. After 3-5 days of culture, viable cells are sorted by FACS, and single cells are seeded into 96-well plates for two additional passages of expansion. Samples are collected for Sanger sequencing to screen successfully constructed cell lines.

Gene Editing Group

Prevention of ferret airway basal cell transplantation PROCEDURE

Before surgery, basal cells (BCs) of recipient ferrets are collected by bronchoscopic brushing, followed by in vitro culture and identification. Prior to the onset of BOS, the cells are injected into recipient ferrets via bronchoscopy. Outcomes including ferret survival rate, body weight, CT imaging, and lung tissue pathology are collected to evaluate the preventive effect of BCs on BOS.

Ferret airway basal cell transplantation group (prevention group)

Transplantation treatment of ferret airway basal cells PROCEDURE

Stable cell lines with target gene knockdown are established in ferret basal cells (BCs). After ferrets develop bronchiolitis obliterans syndrome (BOS), the constructed cell lines are transplanted into recipient ferrets to validate the therapeutic effect of gene-corrected BCs on the disease.

Ferret airway basal cell transplantation group (treatment group)

Eligibility Criteria

• Sex: all
• Healthy Volunteers: Yes
• Age Groups: Child (0-17), Adult (18-64), Older Adult (65+)

You may qualify if:

• Patients who have received allogeneic hematopoietic stem cell transplantation and are diagnosed with bronchiolitis obliterans syndrome

You may not qualify if:

• Bronchoscopy consultation is not suitable for patients who are not suitable for bronchoscopy.

Sponsors & Collaborators

• Haikou Affiliated Hospital of Central South University Xiangya School of Medicine: lead

Study Sites (1)

Haikou Affiliated Hospital of Central South University Xiangya School of Medicine

Haikou, Hainan, 570208, China

Location

MeSH Terms

Conditions

Bronchiolitis Obliterans Syndrome

Interventions

Cloning, Organism; Gene Editing

Condition Hierarchy (Ancestors)

Organizing Pneumonia; Bronchiolitis Obliterans; Bronchiolitis; Bronchitis; Bronchial Diseases; Respiratory Tract Diseases; Lung Diseases, Obstructive; Lung Diseases; Graft vs Host Disease; Immune System Diseases

Intervention Hierarchy (Ancestors)

Genetic Techniques; Investigative Techniques; Reproductive Techniques; Genetic Engineering

Study Officials

• Xiaoyang Yang, DM

  Department of Hematology, Haikou People's Hospital

  PRINCIPAL INVESTIGATOR

Study Design

• Study Type: interventional
• Phase: not applicable
• Allocation: NON RANDOMIZED
• Masking: NONE
• Purpose: SUPPORTIVE CARE
• Intervention Model: PARALLEL
• Sponsor Type: OTHER
• Responsible Party: SPONSOR

Model Details: In this experiment, bronchoscopic brushing was used to collect epithelial mucosal tissues from the diseased lung area and the relatively healthy lung lobe area of the patients. When conditions permit, alveolar lavage fluid specimens of the patients were collected simultaneously to isolate distal small airway stem cells. Allelic samples from healthy volunteers with no obvious airway abnormalities were used as parallel controls. The collected samples were digested with tissue collagenase and then used for the culture of airway BCS.

Study Record Dates

• First Submitted: May 17, 2025
• First Posted: June 12, 2025
• Study Start: January 1, 2026
• Primary Completion (Estimated): June 30, 2027
• Study Completion (Estimated): June 30, 2028
• Last Updated: June 12, 2025

Record last verified: 2025-06

Data Sharing

• IPD Sharing: Will not share

Locations

CN (1)