Towards gene therapy for IPEX syndrome.

Immune dysregulation polyendocrinopathy enteropathy X linked (IPEX) syndrome is an uncurable disease of the immune system, with immune dysregulation that is caused by mutations in FOXP3. Current treatment options, such as pharmacological immune suppression and allogeneic hematopoietic stem cell transplantation, have been beneficial but present limitations, and their life-long consequences are ill-defined. Other similar blood monogenic diseases have been successfully treated using gene transfer in autologous patient cells, thus providing an effective and less invasive therapeutic. Development of gene therapy for patients with IPEX is particularly challenging because successful strategies must restore the complex expression profile of the transcription factor FOXP3, ensuring it is tightly regulated and its cell subset-specific roles are maintained. This review summarizes current efforts toward achieving gene therapy to treat immune dysregulation in IPEX patients.

Design of experiments as a decision tool for cell therapy manufacturing.

BACKGROUND AIMS: Cell therapies are costlier to manufacture than small molecules and protein therapeutics because they require multiple manipulations and are often produced in an autologous manner. Strategies to lower the cost of goods to produce a cell therapy could make a significant impact on its total cost. METHODS: Borrowing from the field of bioprocess development, the authors took a design of experiments (DoE)-based approach to understanding the manufacture of a cell therapy product in pre-clinical development, analyzing main cost factors in the production process. The cells used for these studies were autologous CD4+ T lymphocytes gene-edited using CRISPR/Cas9 and recombinant adeno-associated virus (AAV) to restore normal FOXP3 gene expression as a prospective investigational product for patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. RESULTS: Using gene editing efficiency as the response variable, an initial screen was conducted for other variables that could influence the editing frequency. The multiplicity of infection (MOI) of AAV and amount of single guide RNA (sgRNA) were the significant factors used for the optimization step to generate a response contour plot. Cost analysis was done for multiple points in the design space to find cost drivers that could be reduced. For the range of values tested (50 000-750 000 vg/cell AAV and 0.8-4 µg sgRNA), editing with the highest MOI and sgRNA yielded the best gene editing frequency. However, cost analysis showed the optimal solution was gene editing at 193 000 vg/cell AAV and 1.78 µg sgRNA. CONCLUSIONS: The authors used DoE to define key factors affecting the gene editing process for a potential investigational therapeutic, providing a novel and faster data-based approach to understanding factors driving complex biological processes. This approach could be applied in process development and aid in achieving more robust strategies for the manufacture of cellular therapeutics.

Identification of unstable regulatory and autoreactive effector T cells that are expanded in patients with FOXP3 mutations.

Studies of the monogenic autoimmune disease immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) have elucidated the essential function of the transcription factor FOXP3 and thymic-derived regulatory T cells (Tregs) in controlling peripheral tolerance. However, the presence and the source of autoreactive T cells in IPEX remain undetermined. Here, we investigated how FOXP3 deficiency affects the T cell receptor (TCR) repertoire and Treg stability in vivo and compared T cell abnormalities in patients with IPEX with those in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED). To study Tregs independently of their phenotype and to analyze T cell autoreactivity, we combined Treg-specific demethylation region analyses, single-cell multiomic profiling, and bulk TCR sequencing. We found that patients with IPEX, unlike patients with APECED, have expanded autoreactive T cells originating from both autoreactive effector T cells (Teffs) and Tregs. In addition, a fraction of the expanded Tregs from patients with IPEX lost their phenotypic and functional markers, including CD25 and FOXP3. Functional experiments with CRISPR-Cas9-mediated FOXP3 knockout Tregs and Tregs from patients with IPEX indicated that the patients' Tregs gain a TH2-skewed Teff-like function, which is consistent with immune dysregulation observed in these patients. Analyses of FOXP3 mutation-carrier mothers and a patient with IPEX after hematopoietic stem cell transplantation indicated that Tregs expressing nonmutated FOXP3 prevent the accumulation of autoreactive Teffs and unstable Tregs. These findings could be directly used for diagnostic and prognostic purposes and for monitoring the effects of immunomodulatory treatments.

Co-Expression of FOXP3FL and FOXP3Δ2 Isoforms Is Required for Optimal Treg-Like Cell Phenotypes and Suppressive Function.

FOXP3 is the master transcription factor in both murine and human FOXP3+ regulatory T cells (Tregs), a T-cell subset with a central role in controlling immune responses. Loss of the functional Foxp3 protein in scurfy mice leads to acute early-onset lethal lymphoproliferation. Similarly, pathogenic FOXP3 mutations in humans lead to immunodysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome, which are characterized by systemic autoimmunity that typically begins in the first year of life. However, although pathogenic FOXP3 mutations lead to overlapping phenotypic consequences in both systems, FOXP3 in human Tregs, but not mouse, is expressed as two predominant isoforms, the full length (FOXP3FL) and the alternatively spliced isoform, delta 2 (FOXP3Δ2). Here, using CRISPR/Cas9 to generate FOXP3 knockout CD4+ T cells (FOXP3KOGFP CD4+ T cells), we restore the expression of each isoform by lentiviral gene transfer to delineate their functional roles in human Tregs. When compared to FOXP3FL or FOXP3Δ2 alone, or double transduction of the same isoform, co-expression of FOXP3FL and FOXP3Δ2 induced the highest overall FOXP3 protein expression in FOXP3KOGFP CD4+ T cells. This condition, in turn, led to optimal acquisition of Treg-like cell phenotypes including downregulation of cytokines, such as IL-17, and increased suppressive function. Our data confirm that co-expression of FOXP3FL and FOXP3Δ2 leads to optimal Treg-like cell function and supports the need to maintain the expression of both when engineering therapeutics designed to restore FOXP3 function in otherwise deficient cells.

BHLHE40 Regulates IL-10 and IFN-γ Production in T Cells but Does Not Interfere With Human Type 1 Regulatory T Cell Differentiation.

Type 1 regulatory T (Tr1) cells are subset of peripherally induced antigen-specific regulatory T cells. IL-10 signaling has been shown to be indispensable for polarization and function of Tr1 cells. However, the transcriptional machinery underlying human Tr1 cell differentiation and function is not yet elucidated. To this end, we performed RNA sequencing on ex vivo human CD49b+LAG3+ Tr1 cells. We identified the transcription factor, BHLHE40, to be highly expressed in Tr1 cells. Even though Tr1 cells characteristically produce high levels of IL-10, we found that BHLHE40 represses IL-10 and increases IFN-γ secretion in naïve CD4+ T cells. Through CRISPR/Cas9-mediated knockout, we determined that IL10 significantly increased in the sgBHLHE40-edited cells and BHLHE40 is dispensable for naïve CD4+ T cells to differentiate into Tr1 cells in vitro. Interestingly, BHLHE40 overexpression induces the surface expression of CD49b and LAG3, co-expressed surface molecules attributed to Tr1 cells, but promotes IFN-γ production. Our findings uncover a novel mechanism whereby BHLHE40 acts as a regulator of IL-10 and IFN-γ in human CD4+ T cells.

Review: In vitro generation of red blood cells for transfusion medicine: Progress, prospects and challenges.

In vitro generation of red blood cells (RBCs) has the potential to circumvent the shortfalls in global demand for blood for transfusion applications. The conventional approach for RBC generation has been from differentiation of hematopoietic stem cells (HSCs) derived from cord blood, adult bone marrow or peripheral blood. More recently, RBCs have been generated from human induced pluripotent stem cells (hiPSCs) as well as from immortalized adult erythroid progenitors. In this review, we highlight the recent advances to RBC generation from these different approaches and discuss the challenges and new strategies that can potentially make large-scale in vitro generation of RBCs a feasible approach.

Defined Serum-Free Medium for Bioreactor Culture of an Immortalized Human Erythroblast Cell Line.

Anticipated shortages in donated blood supply have prompted investigation of alternative approaches for in vitro production of red blood cells (RBCs), such as expansion of conditional immortalization erythroid progenitors. However, there is a bioprocessing challenge wherein factors promoting maximal cell expansion and growth-limiting inhibitory factors are yet to be investigated. The authors use an erythroblast cell line (ImEry) derived from immortalizing CD71+CD235a+ erythroblast from adult peripheral blood for optimization of expansion culture conditions. Design of experiments (DOE) is used in media formulation to explore relationships and interactive effects between factors which affect cell expansion. Our in-house optimized medium formulation produced significantly higher cell densities (3.62 ± 0.055) × 106 cells mL-1 , n = 3) compared to commercial formulations (2.07 ± 0.055) × 106 cells mL-1 , n = 3; at 209 h culture). Culture media costs per unit of blood is shown to have a 2.96-3.09 times cost reduction. As a proof of principle for scale up, ImEry are expanded in a half-liter stirred-bioreactor under controlled settings. Growth characteristics, metabolic, and molecular profile of the cells are evaluated. ImEry has identical O2 binding capacity to adult erythroblasts. Amino acid supplementation results in further yield improvements. The study serves as a first step for scaling up erythroblast expansion in controlled bioreactors.

Decellularized material as scaffolds for tissue engineering studies in long gap esophageal atresia.

INTRODUCTION: Esophageal atresia refers to an anomaly in foetal development in which the esophagus terminates in a blind end. Whilst surgical correction is achievable in most patients, when a long gap is present it still represents a major challenge associated with higher morbidity and mortality. In this context, tissue engineering could represent a successful alternative to restore oesophageal function and structure. Naturally derived biomaterials made of decellularized tissues retain native extracellular matrix architecture and composition, providing a suitable bed for the anchorage and growth of relevant cell types. Areas covered: This review outlines the various strategies and challenges in esophageal tissue engineering, highlighting the evolution of ideas in the development of decellularized scaffolds for clinical use. It explores the interplay between clinical needs, ethical dilemmas, and manufacturing challenges in the development of a tissue engineered decellularized scaffold for oesophageal atresia. Expert opinion: Current progress on oesophageal tissue engineering has enabled effective repair of patch defects, whilst the development of a full circumferential construct remains a challenge. Despite the different approaches available and the improvements achieved, a gold standard for fully functional tissue engineered oesophageal constructs has not been defined yet.