Abstract

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.

Highlights

  • IntroductionBackgroundSCID-X1 CD34+ nt RNP (WT Cas). SCID-X1 CD34+ nt RNP (HiFi Cas9)94.1% Not sequenced Background Not sequenced Not sequenced BackgroundSCID-X1 CD34+19 nt RNP (HiFi Cas9)97.6% Not sequenced Background Not sequenced Not Sequenced BackgroundaExpression determined by www.biogps.org and Gene Expression CommonsTo demonstrate that the genome edited IL-2R is permissive for proliferation upon engagement of IL-2 cytokine, we quantified the levels of proliferation of IL2RG complementary DNA (cDNA) targeted T-cell following T-cell receptor (TCR) stimulation

  • BackgroundSCID-X1 CD34+ 19 nt RNP (WT Cas9)

  • There have been a number of proof-of-concept genome editing (GE) studies to explore the feasibility and safety of using a HR-mediated approach to correcting pathologic mutations in the IL2RG gene as a path to developing an auto-hematopoietic stem and progenitor cells (HSPCs)-based therapy for SCIDX119,20,26,50–54

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Summary

Introduction

BackgroundSCID-X1 CD34+ nt RNP (WT Cas). SCID-X1 CD34+ nt RNP (HiFi Cas9)94.1% Not sequenced Background Not sequenced Not sequenced BackgroundSCID-X1 CD34+19 nt RNP (HiFi Cas9)97.6% Not sequenced Background Not sequenced Not Sequenced BackgroundaExpression determined by www.biogps.org and Gene Expression CommonsTo demonstrate that the genome edited IL-2R is permissive for proliferation upon engagement of IL-2 cytokine, we quantified the levels of proliferation of IL2RG cDNA targeted T-cell following T-cell receptor (TCR) stimulation. SCID-X1 CD34+ 19 nt RNP (WT Cas). To demonstrate that the genome edited IL-2R is permissive for proliferation upon engagement of IL-2 cytokine, we quantified the levels of proliferation of IL2RG cDNA targeted T-cell following T-cell receptor (TCR) stimulation. A carboxyfluorescein succinimidyl ester (CFSE) dilution assay was used to measure whether targeted insertion of the codon-optimized cDNA could support T-cell proliferation. We observed similar proliferation profile in tNGFR+ T cells (marking cells in which the IL2RG cDNA had been KI) compared with mock-targeted cells (Fig. 5e). Our data demonstrate that the genomic integration of an IL2RG codon diverged cDNA at the start site of the endogenous locus preserves normal signaling and proliferation of human T cells

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