The respiratory system of the human body includes airways, two lungs and the respiratory muscles. The main function of the lungs is respiration, the process of oxygen from incoming air entering the blood, and carbon dioxide, a waste gas from the metabolism of food leaving the blood. With each breath, the lungs add fresh oxygen to the blood, which then carries it to the cells that need it to function properly. Doing this vital job, the lungs are the essential breathing organ of human beings as well as other mammals.

Unfortunately, there is a large number of lung diseases that are genetically inherited or acquired during development. To fully understand causes of diseases of the respiratory system in respect of the development of novel therapies, the knowledge about embryonic and fetal lung development is indispensable and of major interest.

The development of the lung is orchestrated by a series of events that include formation and growth of the major airway structures as well as the differentiation into the diverse cell types finally ensuring the establishment of a fully functional breathing organ. In general, pulmonary development can be separated into division and branching of the bronchi (branching), the multiplication of airspaces by partitioning (septation), the formation of alveoli (alveolarization; figure 1), and vascular development (lung vascularisation).


Starting at around day 26 after gestation, the entire development of the lung extends from embryonic period through the fetal period up to birth and even afterwards when maturation continues, finally leading to the definitive structure of the lung (figure 2). In contrast to other organs, lung function is unnecessary for intrauterine existence, however, the lungs must be developed to such an extent that they are immediately ready to function following birth.


 

Rare lung diseases

Representing the essential breathing organ of the human body, the proper function of the lung has to be ensured throughout lifetime. However, there is a variety of diseases affecting the respiratory system including common obstructive disorders like asthma, emphysema, chronic obstructive pulmonary disease (COPD), as well as alpha-1-antitrypsin deficiency, and cystic fibrosis. Thereby, lung diseases are amongst the fastest increasing disease entities worldwide in terms of morbidity and mortality.
Major therapeutic breakthroughs still remain elusive, largely due to wide diversity within patient populations and failing diagnosis due to lacking information about causes of disease. Hence, at present lung transplantation represents the only effective therapy in end-stage lung disease, although its feasibility is limited to very few patients indeed.

Pulmonary disorders like alpha-1-antitrypsin deficiency, cystic fibrosis, and surfactant deficiencies belong to the rare lung diseases. In Europe, a rare disease is defined as one that affects fewer than one out of 2,000 people. However, there are more than four million people in Germany suffering from rare disorders of these there are more than 6,000.

Alpha-1-antitrypsin deficiency (AATD) is a genetic disorder caused by defective production of alpha-1-antitrypsin, the major serum and critical lung inhibitor of serine proteases, particularly neutrophil elastase. Inadequately controlled enzyme activity by neutrophil elastase has been implicated as a cause of tissue destruction in the lung, resulting in emphysema and bronchial disease, which are the two major features of COPD. To date, there are only few therapeutic options for patients with this rare disease. The other disease manifestation affects the liver where missfolded protein results in cellular damage and the development of cirrhosis. For the liver diseases, no therapeutic option is available. Lung disease is equivalent to an early form of COPD and requires standard therapy. However, only few patients qualify for substitution of the protein, and a therapy that has not been evaluated in blinded clinical trials and is quiet costly demanding for an alternative strategy.

Cystic fibrosis (CF) is a complex multi-organ disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. As a rare fatal hereditary disease CF is affecting between 1:2,000 to 1:3,000 newborns in Caucasian populations in Western Europe and Northern America. More than 95% of and mortality in CF is caused by a chronic lung disease characterized by airway mucus plugging, chronic bacterial infection and inflammation, and ultimately death due to respiratory failure caused by progressive destruction of the lung. CF lung disease is caused by defects in vectorial ion transport. Interestingly, CFTR mutants lacking the F508 residue (ΔF508) were eliminated by chaperones and ubiquitination as part of a peripheral protein quality-control system. CFTR acts as a cAMP-regulated Cl- channel and regulator of epithelial Na+ channels (ENaC) and plays a critical role in adequate hydration of airway surfaces. In CF airways, deficient Cl- secretion and increased ENaC-mediated Na+ absorption cause airway surface liquid (ASL) volume depletion, which results in adhesion of dehydrated mucus and impaired mucociliary clearance producing progressive lung disease. To date, no effective treatment is available that targets CF at its root cause. Previous gene therapy efforts failed to correct CFTR function in the lung due to low transfection efficiency, transient CFTR expression and immune responses triggered by gene therapy vectors upon repeated application. Therefore, there is a need to develop new strategies to correct CFTR function and deficient airway ion transport in the lungs of CF patients.

Surfactant deficiencies (SF) is a syndrome in premature infants either caused by developmental insufficiency of surfactant production (see figure 1) and structural immaturity in the lungs or resulting from a genetic defect affecting the production of surfactant associated proteins.
Pulmonary surfactant, a mixture of phospholipids and proteins synthesized, packaged and secreted by alveolar type II cells, lowers surface tension and prevents atelectasis at end-expiration. Genetic abnormalities of surfactant function, specifically mutations in the surfactant protein B (SP-B), surfactant protein C (SP-C) or the ATP binding cassette family member A3 (ABCA3) genes account for an increasing number of formerly idiopathic pediatric and adult interstitial lung disease (ILD). Lung biopsies of young children with these genetic abnormalities consistently show the histopathology of congenital pulmonary alveolar proteinosis present in either lethal disease in newborns or in ILD in older infants and children. SP-B deficiency is an autosomal recessive disorder that occurs in approximately 1 per million live births and has been recognized in diverse racial and ethnic groups. ABCA3 deficiencies are probably more predominant contributors to ILD in infancy, but the frequency of this recessive mutation as the underlying cause is still unknown. To date, no specific treatment for these diseases is available. Exogenous replacement therapy with surfactant proteins has only lead to short term improvements. Besides compassionate care, only lung transplantation has been successful in short term for infants and children with refractory and progressive respiratory failure due to mutations in SP-B, SP-C or ABCA3. Therefore, there is a need to develop new strategies to correct these inherited surfactant dysfunctions in the lungs of newborns and infants.


National Funding of Rare Diseases

The Federal Ministry for Education and Research (BMBF) has established a national funding programme of rare diseases. Aim of the programme is to promote the development of new strategies in the supply and maintenance of patients, methods of diagnosis, and the generation of effective therapies for the treatment of rare diseases. Currently, 16 networks are funded receiving funds in the amount of 24 Mio. €. (period: 2008-2012).
(Further information: BMBF)

Among Heidelberg and Homburg/Saar, Hannover is one of three sites in Germany that have joined the BMBF network “Cellular Approaches for Rare Pulmonary Diseases” (CARPuD) coordinated by Professor Ulrich Martin, head of the LEBAO. Major research of the partners of the CARPuD consortium focus on the rare lung diseases AATD, CF, and CD as already described in detail previously.


BMBF Network CARPuD - Cellular Approaches for Rare Pulmonary Diseases

Stem cell research offers new opportunities for the treatment of rare and until now incurable lung diseases like alpha-1-antitrypsin deficiency (AATD), cystic fibrosis (CF) or surfactant deficiencies (SD). Generated through reprogramming of adult cells, for the first time, induced pluripotent stem (iPS) cells seem to enable the differentiation of patient-specific cells into respiratory cells, which then can be used for functional replacement of damaged airway cells and deliver normal lung function (figure 3). Moreover, iPS cells might also be useful for ex vivo gene therapy to deliver genes that compensate for the functional defects in the diseased lung.

(3) Generation of patient-specific induced pluripotent stem (iPS) cells

Goal of the partners of the CARPuD network (see below and figure 4) is the development of innovative therapies for the treatment of the rare lung diseases like AATD, CF, and SD, based on the concept to genetically correct the patient-specific gene defect in autologous iPS cells prior to expansion, differentiation and clinical application.
Aim of the overall project of the present first funding period of the CARPuD consortium is the generation of iPS cells, their differentiation into lung epithelial cells, and application for elucidation of cellular and molecular pathomechanisms. Besides, cell therapeutic approaches are developed and evaluated in appropriate animal models.
Generation of iPSCs from transgenic mouse models (CPA, Hannover) is followed by in vitro differentiation into affected cell types in case of AATD (RP1, Homburg/Saar, Hannover), CF (RP2, Heidelberg, Hannover) or SF (RP3, Hannover). Moreover, cells are applied for investigation of underlying disease mechanisms and for evaluation of iPS cell-based transplantation strategies by abovementioned partners.

Whereas work schedules provided for the first funding period still are predominantly in the field of basic research, projects of a potential second funding period will concentrate on the application of differentiated lung epithelial cells for advanced studies on cellular disease mechanisms and development of improved (stem) cell-based transplantation strategies for the respective lung diseases. Therefore, patient-specific human iPS cells from patients with abovementioned rare lung diseases will be established, to further improve and scale-up established differentiation protocols and to adapt these protocols from murine iPS cells to human iPS cells. In close collaboration with our clinical partners of the future German Lung Center (coordination: T. Welte, Hannover Medical School) a patients’ fibroblast bank is planned to be established to enable the generation and evaluation of a variety of patient-specific stem cells. These joint activities finally may provide the basis for first pre-clinical large animal experiments and clinical phase-1 studies with the perspective to be transferred to general clinical practice in the future.

CPA serves as a service platform for the generation of iPS cells from murine models of rare genetic pulmonary diseases like AATD, CF, SD, and others, providing standardized stem cell resources to all subprojects of the CARPuD consortium. Beyond that, human iPS cells are generated from patients suffering from the respective conditions. Focusing on the clinical application of iPS cell-based treatments, aim of a potential second funding period will be the establishment of genetic modification strategies to correct or modulate the genetic defects in the respective cell lines. Since the use of patient-specific iPS cells in pre-clinical and clinical studies will require large amounts of cells, we are also going to implement large scale cultivation techniques already available in our consortium. I close collaboration with the planned biomaterial bank project (potential 2nd funding period), we aim for the identification of further co-factors involved in disease-course of AATD, CF, and SD through analyzing genotype – phenotype relations, offering a versatile approach to analyze individual factors on the disease characteristics of the iPS cell-derivatives in comparison to lung tissues from patients’ explants after lung transplantation.

AATD is caused by a genetic defect of the SERPINA1 gene that results in liver damage due to the accumulation of misfolded protein in hepatocytes and in lung damage due to the lack of protection from protease activity. The goal of RP1 is to develop a (stem) cell-based therapy that eventually results in repair of the hepatic defect addressing both affected organs. The methodology includes the development of disease-specific stem cells from mouse models and patients to evaluate the novel therapeutic approach by gene therapy and cell transplantation in preclinical models.
We will perform animal experiments to demonstrate that endogenousely expressed AAT can protect the lung from smoke-induced damage and we are investigating the repopulation capacity of wildtype hepatocytes in livers of animals that express Z-type AAT (PiZ mice). To develop a more comprehensive model of AATD, we are also generating a conditional knockout of one of the major mouse AAT genes.
In particular, the generation of iPS cells from patients - provided by partners from CPA - will allow studies of the diseased cell phenotype in vitro an in vivo after transplantation of iPS cell-derived cells into animal models. With these experiments we aim to translate animal research-based achievements into pre-clinical studies for patients suffering from AAT deficiency.
Whereas the lab of M. Ott has established hepatocyte transplantation in mice and humans and the lentiviral modification of isolated hepatocytes prior to transplantation into recipient animals in the meantime, during a potential next funding period, we further want to evaluate the impact of hepatic cell transplantation for correction of the liver phenotype and amelioration of the lung phenotype.
For future use of patient-derived cells as a cellular transplant, genetic correction of hepatic iPS cell-derivatives will be further refined to provide gene-corrected cells for future transplantation approaches.

Since classical gene therapy strategies have not been successful in case of CF due to low transduction efficiencies in airway cells, immune reactions and failure to achieve long term expression in vivo, we aim for an alternative approach on the basis of the development of an innovative cell replacement therapy for the treatment of CF lung disease based on re-transplanting gene-corrected iPS cell-derived airway epithelial (progenitor) cells into patients.
During the first funding period, in vitro protocols for specifically directing murine iPS cells into bronchial epithelial cells, in particular Clara cells (see figure 2), are established followed by bronchial differentiation of human iPS cells. Since murine and human iPS (hiPS) cells can already be easily genetically manipulated, complementary efforts to correct/repair specific disease-causing mutations in the CFTR gene in hiPS cells derived from CF patients are already planned within the scope of a further funding period.

Moreover, we wish to further optimize airway differentiation of murine iPS cells and evaluate functional integration of iPS cell-derived Clara cells in previously established CF mouse model. Established protocol will be adapted to patient-specific human iPS cells, followed by pilot transplantation experiments in immunodeficient SCID mice to assess whether this novel cutting-edge cell therapy approach may allow for functional repopulation of CF lungs with uncompromised autologous bronchial epithelium.

Major focus of RP3 is the generation of type II alveolar epithelial (AT2) cells (see figure 2) derived from iPS cells and their evaluation in a murine disease model. iPS cells from mice lacking expression of surfactant protein B (SP-B) thereby are established within CPA.
Furthermore, a mouse model for intratracheal application of stem cell derivatives is developed to provide a readout for a functional relevant transplantation of AT2 cells. Therapeutic effects of transplantation of normal iPS cell-derived AT2 cells on survival, surfactant production (see figure 1) and lung function will be determined at the end of the first funding period, whereas further research will focus on adaption of established protocols for in vitro alveolar differentiation of human iPS cells. Aiming at the functional engraftment of hiPS cell-derived AT2 cells and in respect of treatments rare hereditary surfactant deficiencies through transplanting gene-corrected autologous AT2 cells, in vivo evaluation of iPS cell-derived AT2 cells finally will be performed in immunodeficient SCID mice.