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

Goodwin M, Lee E, Lakshmanan U, Shipp S, Froessl L, Barzaghi F, Passerini L, Narula M, Sheikali A, Lee CM, Bao G, Bauer CS, Miller HK, Garcia-Lloret M, Butte MJ, Bertaina A, Shah A, Pavel-Dinu M, Hendel A, Porteus M, Roncarolo MG, Bacchetta R.
Source: Science
Publication Date: (2020)
Issue: 6(19): eaaz0571
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
CD34+ cell, human
Species: human
Tissue Origin: blood
Culture Media:
4D-Nucleofector® X-Unit

Tregs were cultured in X-VIVO 15 (Lonza) with 5% human serum from male AB plasma (Sigma-Aldrich), IL-2 (300 U/ml; PeproTech), and 100 nmol rapamycin (STEMCELL Technologies; only added for certain experiments). Teff cells were cultured in X-VIVO 15, 5% human serum, and IL-2 (50 U/ml). The Tregs and Teff cells were cultured at 37°C with 5% CO2 and ambient oxygen levels. Treg-like MT-2 cells were cultured in X-VIVO 15 with 5% human serum and 1% penicillin/streptomycin. K562 cells (American Type Culture Collection) were cultured in RPMI 1640 medium (Thermo Fisher Scientific) with 10% fetal bovine serum (Thermo Fisher Scientific) at 37°C with 5% CO2 and ambient oxygen levels. For all cells, fresh medium was added every 2 to 3 days.

For the initial screen, 2µg of sgRNA/Cas9 plasmid DNA was nucleofected into 1 million K562 cells using the Lonza Nucleofector 2b (program T-016).

The sgRNA was complexed with Cas9 for 10 min at 25°C at an approximate Cas9:sgRNA molar ratio of 1:2.5, using 8 µg of sgRNA and 15 µg of Cas9 per 100 µl of nucleofection solution containing 2.5 × 105 to 1 × 106 cells. After switching to high-fidelity (HiFi) Cas9 (IDT) that showed slightly lower efficiency in parallel experiments, the amount of HiFi Cas9 was increased, and the molar ratio was adjusted to 1:1.8, using 8 µg of sgRNA with 22 µg of HiFi Cas9 per 100 µl of nucleofection solution. The sgRNA/Cas9 complexes (IDT, TriLink BioTechnologies and Synthego) were nucleofected into Tregs and Teff cells after 2 to 3 days of activation using the P3 Primary Cell Nucleofection Kit (Lonza) and the Lonza Nucleofector 4D (program E-0115). For HSPC editing, the cells were nucleofected using the P3 Primary Cell Nucleofection Kit (Lonza) and the Lonza Nucleofector 4D (program DZ-100). 


The prototypical genetic autoimmune disease is immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, a severe pediatric disease with limited treatment options. IPEX syndrome is caused by mutations in the forkhead box protein 3 (FOXP3) gene, which plays a critical role in immune regulation. As a monogenic disease, IPEX is an ideal candidate for a therapeutic approach in which autologous hematopoietic stem and progenitor (HSPC) cells or T cells are gene edited ex vivo and reinfused. Here, we describe a CRISPR-based gene correction permitting regulated expression of FOXP3 protein. We demonstrate that gene editing preserves HSPC differentiation potential, and that edited regulatory and effector T cells maintain their in vitro phenotype and function. Additionally, we show that this strategy is suitable for IPEX patient cells with diverse mutations. These results demonstrate the feasibility of gene correction, which will be instrumental for the development of therapeutic approaches for other genetic autoimmune diseases.