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Characterisation of Thymic Stromal Cell Defects

Supervisors: Professor Adrian Thrasher, Dr Alexandra Kreins

Background:
Athymia, congenital absence of the thymus, is usually fatal in the first two years of life due to T-cell immunodeficiency. Most commonly this problem is associated with complete DiGeorge Syndrome, but it is also found in patients with rare (monogenic) types of Severe Combined Immunodeficiency1. Athymia results from abnormal patterning of the pharyngeal apparatus or impaired development of thymic epithelial cells (TEC) during embryogenesis2. Currently the only definitive treatment for athymic patients is allogeneic thymus transplantation (TT). The TT programme at UCL’s Institute of Child Health and Great Ormond Street Hospital is one of only two programmes worldwide to offer this treatment3. Suboptimal recovery of T-cell immunity and autoimmunity remain frequent complications. Recently, reprogrammed fibroblasts and induced pluripotent stem cells (iPSC) have been used for the generation of thymic epithelial progenitor cells (iTEPC)4, offering new avenues for disease modelling and treatments.

Aims/Objectives:

The general aim of this translational research project is to further characterise thymic stromal cell defects, by investigating how they affect TEC differentiation when recapitulating ontogeny starting from induced pluripotent stem cells (iPSC), thus increasing our knowledge on thymus development.

Objective 1: Using gene editing tools, mutations of athymic patients will be knocked in control iPSC lines, making it possible to study their impact on the different developmental steps of TEC ontogeny in vitro. Objective 2: iPSCs from patients with genetically defined thymic stromal cell defects will be generated. Developmental blocks in TEC ontogeny will be identified and characterised in vitro in comparison with wildtype and gene-edited control iPSC lines, as well as bona fide TEC progenitors5. Objective 3: Functional studies will be conducted on gene-corrected patient-derived iPSC lines, generating preliminary pre-clinical data for future autologous TT treatment modalities, possibly using gene-corrected engineered thymus tissue1,5.

Methods:
Patient recruitment and material: This project will have access to a unique cohort of athymic patients with a variety of thymic stromal cell defects. Stored patient materiel will be used, including primary skin fibroblasts which will be re-programmed into iPSCs. Targeted iTEPC differentiation: iPSCs will be differentiated into definitive endoderm and subsequently iTEPCs adapting existing protocols4. Gene-editing and ontogenetic characterisation: In addition to routine quality control of iTEPC differentiation by flow cytometry, comprehensive epigenetic and transcriptomic studies will be undertaken. CRISPR/Cas9 gene-editing will facilitate the study of the impact of disease-causing mutations on TEC ontogeny.

References:
1. Kreins AY, Bonfanti P, Davies EG. Current and future therapeutic approaches for thymic stromal cell defects. Front Immunol 2021; 12:655354.
2. Kreins AY, Maio S, Dhalla F. Inborn errors of thymic stromal cell development and function. Semin Immunopathol 2021; 43(1):85-100.
3. Davies EG et al. Thymus transplantation for complete DiGeorge syndrome: European experience. J Allergy Clin Immunol 2017; 140:1660-70.
4. Chhatta A et al. De novo generation of a functional human thymus from induced pluripotent stem cells. J Allergy Clin Immunol 2019; 144(5):1416-1419.
5. Campinoti S et al. Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds. Nat Commun 2020; 11(1):6372.