Gene Correction for SCID-X1 In Long-Term Hematopoietic Stem Cells


Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34 + HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl. ©2019 The Author(s).


Includes supplementary material


Cd28 Antigens, Antigens, CD34, Doxycycline, DNA Probes, Interleukin-2, Interleukin 2 Receptor Gamma Chain, Mouse, Interleukin-7, Nucleoproteins, STAT5 protein, T cells—Receptors, Adulthood, Alleles, Bioinformatics, Cell differentiation, Cell separation, Cell proliferation, Stem cells, CRISPR-associated protein 9, Electroporation, Exons, Females, Flow cytometry, Gene editing, Gene expression, Gene frequency, Gene targeting, Hematopoiesis, Hematopoietic stem cells, High performance liquid chromatography, Humans, Human cell culture, Karyotypes, Lymphopoiesis, Males, Mice, Mutation (Biology), Phenotype, Polymerase chain reaction, Signal transduction, Codon, Initiator, Targeted gene repair, Toxicity testing, Transplantation, Heterologous

CIRM (PC1-08111), NIH/NIAID (R01 AI097320-01), Cancer Prevention and Research Institute of Texas (RR140081 and RR170721).


CC BY 4.0 (Attribution), ©2019 The Authors