High Efficiency Gene Correction in Hematopoietic Cells by Donor Template-free CRISPR/Cas9 Genome Editing

• Main Author: Sürün, Duran
• Format: Others
• Language: en
• Published: 2018
• Online Access: https://tuprints.ulb.tu-darmstadt.de/7247/7/Dissertation_nach_Disputation.pdf; Sürün, Duran <http://tuprints.ulb.tu-darmstadt.de/view/person/S=FCr=FCn=3ADuran=3A=3A.html> (2018): High Efficiency Gene Correction in Hematopoietic Cells by Donor Template-free CRISPR/Cas9 Genome Editing.Darmstadt, Technische Universität, [Ph.D. Thesis]

A significant fraction of inherited monogenic disorders are caused by patient-specific mutations dispersed over the entire locus of the affected gene. Although correcting these mutations by introducing healthy gene copies into the genome of the diseased cells proved effective in several clinical gene therapy trials and with more advanced vectors safety and efficacy could be improved, insertional mutagenesis and unregulated expression of genes deprived of their endogenous control elements remains a concern when using randomly integrating vectors. As has been shown repeatedly in clinical trials random vector insertions are susceptible to epigenetic silencing and can cause cancer by the activation of adjacent proto-oncogenes. The development of genome editing tools capable of modifying any prespecified genomic sequence with unprecedented accuracy opened up a wide range of new possibilities in gene manipulation including targeted gene repair. In particular, CRISPR/Cas9 system, a prokaryotic adaptive immune system and its swift repurposing for genome editing was widely adopted as the hitherto simplest genome editing tool. In combination with a single guide RNA (sgRNA) the Cas9 endonuclease generates DNA double strand breaks (DSBs) at prespecified genomic loci that are repaired either by homology directed repair (HDR) or nonhomologous end joining (NHEJ). Correction of human disease mutations by this technology has been thus far largely based on homologous recombination requiring an exogenous donor template along with RNA guided (gRNA) Cas9 endonucleases (RGNs). In most applications, RGNs and templates were delivered to the diseased cells by electroporation of several plasmids each expressing one of the functional components needed for targeted gene modification. However, transducing the functional components required for homology directed repair (HDR) on different plasmids and considering that electroporation is quite harmful to the target cells, only a small fraction of the cells survive transfection and even fewer retain all functional components. As a result, the number of gene corrected cells is usually quite low and reduced even further by the inherent bias of the cell's double strand break (DSB) machinery towards NHEJ. This thesis explores the efficiency of gene repair by NHEJ in hematopoietic cells harboring patient specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB) whose inactivation causes chronic granulomatous disease (X-CGD), - a life-threatening immunodeficiency disorder. Although in contrast to HDR, NHEJ is error prone, the present work was based on the theoretical assumption that about, one-third of the insertions/deletions (indels) associated with NHEJ should restore the open reading frame (ORF) disrupted by a particular disease mutation. This would lead to a significant number of ORF reconstitutions of which some, depending on the position and type of the original mutation, should either completely or partially recover protein function. Moreover, donor template free delivery of RGNs on one rather than multiple expression vectors by lentiviral infection was expected to improve gene repair efficiencies and to reduce toxicity of gene transduction. In initial experiments designed to determine the efficiency of gene repair by NHEJ 32D hematopoietic cells expressing four different EGFP reporter transgenes harboring N-terminal frameshift mutations were nucleofected each with Cas9 and corresponding sgRNAs. Consistent with previous genome editing protocols involving transfection, gene repair efficiency was low, ranging from 2.3% to 5.5%. Similar testing was performed in human PLB-985 leukemia cells expressing one copy of a mutationally inactivated EGFP reporter (mEGFP). However, to increase transduction rates and ensure transient RGN expression, the RGNs were delivered by integration defective lentiviruses (IDLVs). Unlike transfection IDLV delivery of RGNs yielded high on-target mutation rates leading to mEGFP repair rates of up to 27%. Collectively, the results demonstrate that mEGFP repair efficiency improved by one order of magnitude after changing the RGN delivery protocol from plasmid nucleofection to IDLV infection. This strategy was tested further in PLB cells harboring bona fide disease mutations. For this, four X-CGD-patient specific CYBB mutations including two frameshift, one nonsense and one missense mutation were individually transduced into CYBB null PLB cells (XCGD-PLB). While subsequent delivery of the corresponding RGNs effectively repaired the frameshift mutations in up to 10% of the treated cells, the repair efficiency of the nonsense and missense mutations was with less than 2% rather ineffective. As about 20 - 25% of most inherited blood disorders are caused by frameshift mutations, the results of this thesis suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from a gene therapy employing personalized, donor-template free RGNs.