DeRavin, S. S. | bioRxiv Science

bioRxiv2019

Aberrant Clonal Hematopoiesis Following Lentiviral Transduction of HSPC in a Rhesus Macaque

Lentiviral vectors (LV) have been used for the delivery of genes into hematopoietic stem and progenitor cells (HSPC) in clinical trials worldwide. LV, in contrast to retroviral vectors, have not been associated with insertion site-associated malignant clonal expansions, and thus have been considered safer. Here, however, we present a case of markedly abnormal dysplastic clonal hematopoiesis impacting the erythroid, myeloid and megakaryocytic lineages in a rhesus macaque transplanted with HSPCs that were transduced with a LV containing a strong retroviral murine stem cell virus (MSCV) constitutive promoter-enhancer in the LTR. 9 insertions were mapped in the abnormal clone, resulting in overexpression and aberrant splicing of several genes of interest, including the cytokine stem cell factor and the transcription factor PLAG1. This case represents the first clear link between a lentiviral insertion-induced clonal expansion and a clinically abnormal transformed phenotype following transduction of normal primate or human HSPC, and are thus concerning, and suggest that strong constitutive promoters should not be included within LV vectors.

molecular biology↗

bioRxiv2018

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. High frequencies of reproducible targeted integration of a wild-type cDNA into the endogenous start codon of a gene in LT-HSCs could provide a robust genome editing approach to cure genetic diseases in which patients have different mutations throughout the gene. We describe a clinically relevant method for correcting X-linked severe combined immunodeficiency (SCID-X1). By using a highly specific and active CRISPR/Cas9-AAV6 based strategy and selection-free approach, we achieve up to 20% genome integration frequencies in LT-HSCs of a full-length IL2RG cDNA at the endogenous start site as demonstrated by serial transplantation and analysis of genome edited human cells eight months following initial transplantation. In addition to high frequencies of functional gene correction in LT-HSCs we observed no 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.

bioengineering↗