Chromosome Transplantation: Correction of the Chronic Granulomatous Disease Defect in Mouse Induced Pluripotent Stem Cells

Abstract

In spite of the progress in gene editing achieved in recent years, a subset of genetic diseases involving structural chromosome abnormalities, including aneuploidies, large deletions and complex rearrangements, cannot be treated with conventional gene therapy approaches. We have previously devised a strategy, dubbed chromosome transplantation (CT), to replace an endogenous mutated chromosome with an exogenous normal one. To establish a proof of principle for our approach, we chose as disease model the chronic granulomatous disease (CGD), an X‐linked severe immunodeficiency due to abnormalities in CYBB (GP91) gene, including large genomic deletions. We corrected the gene defect by CT in induced pluripotent stem cells (iPSCs) from a CGD male mouse model. The Hprt gene of the endogenous X chromosome was inactivated by CRISPR/Cas9 technology thus allowing the exploitation of the hypoxanthine–aminopterin–thymidine selection system to introduce a normal donor X chromosome by microcell‐mediated chromosome transfer. X‐transplanted clones were obtained, and diploid XY clones which spontaneously lost the endogenous X chromosome were isolated. These cells were differentiated toward the myeloid lineage, and functional granulocytes producing GP91 protein were obtained. We propose the CT approach to correct iPSCs from patients affected by other X‐linked diseases with large deletions, whose treatment is still unsatisfactory. Stem Cells 2019;37:876–887

Chromosome Transplantation: Opportunities and Limitations

“…Indeed, it has already been demonstrated that these patients can benefit from granulocyte infusion from unrelated donors [50]. We were able to show that, after Hprt gene inactivation in iPSCs derived from a mouse model of this disease and fusion with a donor cell line, normal diploid clones, able to differentiate to functionally corrected granulocytes, could be easily obtained [31]. In this way, a theoretically infinite number of autologous granulocytes could be produced.…”

“…Further studies have shown that by the CRISPR-mediated HPRT gene inactivation approach used in mice [31], human-chromosome-transplanted iPSCs can be obtained from patients affected by other diseases mapping to the X chromosome such as Duchenne muscular dystrophy (DMD). DMD is one of the best candidates for CT since its gene is larger than 2 Mb and it is often due to gross deletions that cannot be treated by conventional gene therapy.…”

“…In the last few years, it became clear that this phenomenon can also occur in vitro, where the rescue of a diploid state can be easily detected and isolated. This has been the basis of the isolation of rescued diploid clones in both mouse and human cells [22,30,31], which can be achieved by cytogenetic examination or PCR screening (for example, it is possible to identify the presence of the wild type and the absence of the mutated HPRT gene corresponding to the exogenous and endogenous X chromosome, respectively). Recently, Murakami and coworkers (see below) exploited the natural tendency to lose the Y chromosome to generate 39,X0 mouse iPSCs as a first step to produce 40,XX normal female cells from male cells [32].…”

Hematopoietic Cells from Pluripotent Stem Cells: Hope and Promise for the Treatment of Inherited Blood Disorders

“…We used this type of approach on human and murine pluripotent stem cells to address treatment of different type of diseases [20,21], including hematological disorders. In detail, we replaced the defective X chromosome in iPSCs derived from a murine model of CGD with a WT X chromosome obtained from a healthy donor [58]. The endogenous defective X chromosome was previously engineered through the CRISPR/Cas9 system to obtain Hprt inactivation, as a mean to discriminate the exogenous X chromosome from the endogenous defective one.…”

Chromosome Transplantation: A Possible Approach to Treat Human X-linked Disorders

“…We showed the feasibility of this approach in mice by performing CT in Hprt-deficient embryonic stem cells (ESCs), 17 a model for the human Lesch-Nyhan (LN) disease, and in induced pluripotent stem cells (iPSCs) from a mouse model of chronic granulomatous disease (CGD). 19 However, to our knowledge, human pluripotent stem cells have so far been refractory to CT, although recently an artificial chromosome has been successfully transferred to human iPSCs. 20 In this study, we report the achievement for the first time of CT in human iPSCs using as proof of concept reprogrammed cells from a LN patient carrying a mutation in the X-linked HPRT gene useful for the selection of the corrected cells.…”