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 d AAV6 is an effective donor delivery vector for genome editing in hPSCs d Electroporation of Cas9 RNP prior to AAV6 transduction yields editing up to 90% d The Cas9 RNP/AAV6 method allows for specific modifications ranging from 1 to >3,000 bp d This method yields highly edited cells without selection markers or antibiotics

Differentiating somatic cells are progressively restricted to specialized functions during ontogeny, but they can be experimentally directed to form other cell types, including those with complete embryonic potential. Early nuclear reprogramming methods, such as somatic cell nuclear transfer (SCNT) and cell fusion, posed significant technical hurdles to precise dissection of the regulatory programmes governing cell identity. However, the discovery of reprogramming by ectopic expression of a defined set of transcription factors, known as direct reprogramming, provided a tractable platform to uncover molecular characteristics of cellular specification and differentiation, cell type stability and pluripotency. We discuss the control and maintenance of cellular identity during developmental transitions as they have been studied using direct reprogramming, with an emphasis on transcriptional and epigenetic regulation.

Gene correction in human long-term hematopoietic stem cells (LT-HSCs(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/397463 doi: bioRxiv preprint first posted online Aug. 22, 2018; 2 evidence of abnormal hematopoiesis following transplantation, a functional measure of the lack of genotoxicity. Deep analysis of potential off-target activity detected two sites with low frequency (<0.3%) of off-target mutations. The level of off-target mutations was reduced to below the limit of detection using a high fidelity Cas9.Moreover, karyotype evaluation identified no genomic instability events. We achieved high levels of genome targeting frequencies (median 45%) in CD34 + HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect of patient derived cells both 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.X-linked severe combined immunodeficiency (SCID-X1) is a rare, primary immune deficiency (PID) caused by mutations in the IL2RG gene on the X-chromosome.The gene encodes a shared subunit of the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Without early treatment, affected male infants die in the first year of life from infections. Although allogeneic hematopoietic cell transplant (allo-HCT) is considered the standard of care for SCID-X1, it holds significant risks due to potential incomplete immune reconstitution, graft versus host disease (GvHD) and a decreased survival rate in the absence of an HLA-matched sibling donor 1 . Importantly, because of the selective advantage of lymphoid progenitors expressing normal IL2RG, only a small number of genetically corrected hematopoietic stem and progenitor cells (HSPCs) are needed to reconstitute T-cell immunity. The selective advantage has been demonstrated both by allo-HCT and by rare experiments of nature, in which a spontaneous reversion in a hematopoietic progenitor gives rise to a functional T-cell repertoire in a SCID-X1 patient 2,3 . The rare patients with reversion mutations and patients who received allo-HCT . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/397463 doi: bioRxiv preprint first posted online Aug. 22, 2018; 3 without giving conditioning chemotherapy also highlights the importance of achieving gene correction in long-term hematopoietic stem cells (LT-HSCs) to achieve sustained clinical benefit as patients who do not have corrected LT-HSC engraftment often end up losing a robust and functional T-cell immune system over time.Gene therapy is an alternative therapy to allo-HSCT. Using integrating viral vectors, such as gamma-retroviral and lentiviral vectors, extra copie...

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