CRISPR-mediated genotypic and phenotypic correction of a chronic granulomatous disease mutation in human iPS cells

Abstract

Chronic granulomatous disease (CGD) is a rare genetic disease characterized by severe and persistent childhood infections. It is caused by the lack of an antipathogen oxidative burst, normally performed by phagocytic cells to contain and clear bacterial and fungal growth. Restoration of immune function can be achieved with heterologous bone marrow transplantation; however, autologous bone marrow transplantation would be a preferable option. Thus, a method is required to recapitulate the function of the diseased gene within the patient's own cells. Gene therapy approaches for CGD have employed randomly integrating viruses with concomitant issues of insertional mutagenesis, inaccurate gene dosage, and gene silencing. Here, we explore the potential of the recently described clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 site-specific nuclease system to encourage repair of the endogenous gene by enhancing the levels of homologous recombination. Using induced pluripotent stem cells derived from a CGD patient containing a single intronic mutation in the CYBB gene, we show that footprintless gene editing is a viable option to correct disease mutations. Gene correction results in restoration of oxidative burst function in iPS-derived phagocytes by reintroduction of a previously skipped exon in the cytochrome b-245 heavy chain (CYBB) protein. This study provides proof-of-principle for a gene therapy approach to CGD treatment using CRISPR-Cas9.

Urine-derived induced pluripotent stem cells as a modeling tool to study rare human diseases

“…iPSCs were generated from a patient with mutant CYBB, and the mutations were corrected using a CRISPR-Cas9 site-specific nuclease system (88). Phagocytes derived from these CYBB-corrected iPSCs restored the oxidative burst.…”

Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

“…19 CGD patient-specific iPSCs were successfully differentiated into neutrophils and/or macrophages recapitulating the ROS-deficient phenotype to test new therapeutic strategies. [20][21][22][23][24][25][26] Then, using confocal microscopy and flow cytometry, we demonstrated the capability of liposomes to transport and deliver recombinant cytochrome b 558 to the membrane of X 0 -linked CGD (X 0 -CGD) iPSC-derived macrophages. Finally, the therapeutic potential of the NOX2/p22 phox liposomes was evidenced by the restoration of the NADPH oxidase activity of these ROS-deficient cells using the nitroblue tetrazolium (NBT) chloride test.…”

CRISPR/Cas9-mediated traceless gene correction and activation of the HBB locus in iPSCs with the beta thalassaemia mutation

“…Combining strategies that enhance the preciseness of available molecular biology tools (i.e. CRISPR/Cas9) can be useful in improving the on-target editing rate [145,212] and in achieving highly specific genome engineering [100]. Precisely engineered iPSCs are needed for stem cell-based modelling of genetic disease, and for providing an unlimited source of gene-edited lines for potential use in regenerative medicine [228,229].…”

“…double nickase (Cas9n) such as is used in the DN strategy), which induce DSBs at the on-target site while only single nicks are possible at each gRNA's off-target sites, would be more advantageous for precise DNA modification [96]. Because the on-target editing rate mediated by CRISPR/Cas9 is influenced by various factors that include the strategy design [145,212], it was hypothesised that preventing the iterative cycles of dual Cas9n activity (introduction of DSBs) by either separation of the guide pair that targets the locus of interest [145] or by introducing a synonymous SNP in the PAM sequence [212] would be effective at limiting the on-target mutagenesis using the current approach. The double nickase strategy was explored using two different gRNA pairs in conjunction with a piggyBac transposon-based design for the donor vector.…”

“…To demonstrate the restoration of the wild type phenotype in gene-corrected disease iPSCs, the corrected cells are commonly differentiated into specialised cell types that express the edited gene [77,212]. However, differentiated iPSCs mostly fail to adopt a mature phenotype, and this evidently limits the functional analysis of the corrected allele for genes that are associated with mature phenotypic characteristics.…”