Chromosome transplantation as a novel approach for correcting complex genomic disorders

Abstract

Genomic disorders resulting from large rearrangements of the genome remain an important unsolved issue in gene therapy. Chromosome transplantation, defined as the perfect replacement of an endogenous chromosome with a homologous one, has the potential of curing this kind of disorders. Here we report the first successful case of chromosome transplantation by replacement of an endogenous X chromosome carrying a mutation in the Hprt gene with a normal one in mouse embryonic stem cells (ESCs), correcting the genetic defect. The defect was also corrected by replacing the Y chromosome with an X chromosome. Chromosome transplanted clones maintained in vitro and in vivo features of stemness and contributed to chimera formation. Genome integrity was confirmed by cytogenetic and molecular genome analysis. The approach here proposed, with some modifications, might be used to cure various disorders due to other X chromosome aberrations in induced pluripotent stem (iPS) cells derived from affected patients.

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

“…To obtain the correction of CGD genetic defect in the established mouse CGD‐iPSC lines, we applied the CT protocol (Fig. ) recently set up in mouse ESCs . As recipient cell line, we used the CGD‐33 clone in which we inactivated the endogenous Hprt gene through CRISPR technology , by targeting exon 3 with a specific gRNA (gRNA‐Ex3‐ Hprt ), to take advantage of the HAT/6‐TG positive/negative selection system.…”

“…They include aneuploidies, large deletions, inversions, and defects due to large triplet expansions that cause "genomic disorders" [2]. Recently, the attempt to treat these diseases have established the new field of "chromosome or genomic therapy" [1,[3][4][5][6].…”

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

“…Classical MMCT was performed as previously described, with some modifications. 17,22 After centrifugation at 160 Â g, 1 mL of a prewarmed solution of 50% PEG 1500 (Roche, Mannheim, Germany) was poured onto the cell pellet during 1 min, followed by extensive washing in serum-free RPMI 1640. 2.5 Â 10 6 iPSCs were plated in Matrigel in hESC-qualified matrix p60 dishes, and after 48 h 1Â HAT selection was added.…”

“…In previous work, we have introduced the concept of chromosome transplantation (CT) as a means to achieve correction in the selected cases that cannot benefit by classical gene therapy methods. 17 CT refers to the precise substitution of an endogenous defective chromosome with a normal exogenous one. CT generates euploid cells and is different from chromosome transfer in which the addition of one or a few chromosomes gives rise to aneuploid cells, which cannot be of clinical utility.…”

“…CT is a new approach that is potentially useful in the new field of "genomic or chromosome therapy," 18 an area that has been long neglected. 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.…”