Ectopic FOXP3 Expression Preserves Primitive Features Of Human Hematopoietic Stem Cells While Impairing Functional T Cell Differentiation

Abstract

FOXP3 is the transcription factor ruling regulatory T cell function and maintenance of peripheral immune tolerance, and mutations in its coding gene causes IPEX autoimmune syndrome. FOXP3 is also a cell-cycle inhibitor and onco-suppressor in different cell types. In this work, we investigate the effect of ectopic FOXP3 expression on HSC differentiation and we challenged this approach as a possible HSC-based gene therapy for IPEX. FOXP3-expressing HSC showed reduced proliferation ability and increased maintenance of primitive markers in vitro in both liquid and OP9-ΔL1 co-cultures. When transplanted into immunodeficient mice, FOXP3-expressing HSC showed significantly enhanced engraftment ability. This was due to a pronounced increase in the frequency of repopulating cells, as assessed by extreme limiting dilution assay. Likely underlying the increased repopulating ability, FOXP3 expressing HSC showed significantly enhanced expression of genes controlling stemness features. However, peripheral T cells developed in the FOXP3-humanized mice were quantitatively reduced and hyporesponsive to cytokine and polyclonal stimulation. Our findings reveal unpredicted effects of FOXP3 in the biology of HSC and may provide new tools to manipulate primitive features in HSC for clinical applications. Moreover, they formally prove the need of preserving endogenous FOXP3 regulation for an HSC-based gene therapy approach for IPEX syndrome.

Gene therapy targeting haematopoietic stem cells for inherited diseases: progress and challenges

“…Relative to gene addition strategies, these editing approaches guarantee the endogenous, physiologically regulated expression of the transgene. This is particularly important when the elements that regulate expression of the transgene have not been characterized, the gene or its regulatory elements are too large to fit into high-titre lentiviral vectors or restricted or tightly regulated transgene expression is desirable (for example, in β-haemoglobinopathies, immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome 142 , Artemisdeficient severe combined immunodeficiency (ART-SCID) 143 , interleukin-7 receptor subunit-α-deficient SCID (Il7RA-SCID) 144 and chronic granulomatous disease (CGD) 145,146 ). It is noteworthy that the use of restricted promoters to drive transgene expression with lentiviral vectors can sometimes result in near-physiological expression patterns 147,148 , although several copies of the vector may be required.…”

CRISPR-based gene editing enables FOXP3 gene repair in IPEX patient cells

“…However, this approach does not address other cell types, such as T eff cells, that also contribute to the IPEX pathology. An additional limitation of this approach is that it cannot be used on long-term repopulating hematopoietic stem and progenitor cells (HSPCs) because of the adverse effects of FOXP3 overexpression on stem cell proliferation and differentiation (18). For the development of a successful FOXP3 gene therapy using HSPCs, it is necessary to achieve constitutive expression of FOXP3 in the T reg compartment to restore suppressive function without having FOXP3 overexpression perturb the proliferation and function of HSPCs or T eff cells.…”

Gene Therapy for Primary Immunodeficiency

“… 138 Correction at the level of HSC would provide a longer-lasting therapy, however, studies investigating this approach have noted that constitutive FOXP3 expression in HSC (where it is not usually expressed) had adverse effects on T cell differentiation and hematopoiesis. 139 A recent study aiming to abrogate this effect by replicating the endogenous expression profile by harnessing 3 regulatory elements, the FOXP3 promoter and the 3′UTR (untranslated region) to regulate transgene expression, has shown promising results in vivo. 140 Another recent study using gene editing tools to place FOXP3 cDNA under control of its native promoter reported partial correction of FOXP3 expression and suppressive function restored to within the lower range of healthy control cells, indicating this methodology could provide an alternative approach for IPEX patients.…”