Complete Genetic Correction of iPS Cells From Duchenne Muscular Dystrophy

Abstract

Human artificial chromosome (HAC) has several advantages as a gene therapy vector, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including the regulatory elements. Induced pluripotent stem (iPS) cells have great potential for gene therapy, as such cells can be generated from the individual's own tissues, and when reintroduced can contribute to the specialized function of any tissue. As a proof of concept, we show herein the complete correction of a genetic deficiency in iPS cells derived from Duchenne muscular dystrophy (DMD) model (mdx) mice and a human DMD patient using a HAC with a complete genomic dystrophin sequence (DYS-HAC). Deletion or mutation of dystrophin in iPS cells was corrected by transferring the DYS-HAC via microcell-mediated chromosome transfer (MMCT). DMD patient- and mdx-specific iPS cells with the DYS-HAC gave rise to differentiation of three germ layers in the teratoma, and human dystrophin expression was detected in muscle-like tissues. Furthermore, chimeric mice from mdx-iPS (DYS-HAC) cells were produced and DYS-HAC was detected in all tissues examined, with tissue-specific expression of dystrophin. Therefore, the combination of patient-specific iPS cells and HAC-containing defective genes represents a powerful tool for gene and cell therapies.

Stable maintenance of de novo assembled human artificial chromosomes in embryonic stem cells and their differentiated progeny in mice

“…5 Importantly, this HAC has demonstrated its utility as a high capacity gene therapy vector in mouse models of muscular dystrophies. 3,[6][7][8] This HAC has also been successfully used to deliver reprogramming factors, but its subsequent removal from de novo generated iPS cells is quite problematic and relies on spontaneous loss during mitotic divisions, which is extremely rare. 9 A novel, truly artificial HAC has recently come to the fore as a highly promising vector system.…”

“…Most characterized HACs have been generated by a topdown approach involving truncation of various human chromosomes, 2,3 which, strictly speaking, makes them modified natural chromosomes (mini-chromosomes) rather than artificial ones. All these HACs are maintained as episomes and are mitotically stable.…”

Integration-free and stable expression of FVIII using a human artificial chromosome

“…The use of currently available viral vector systems may be complemented by the use of human artificial chromosomes (HAC) because HAC replicates and segregates independently from host chromosomes as a minichromosome. [2][3][4][5][6] HAC can be transferred into different cell lines by microcell-mediated chromosome transfer. A HAC vector derived from human chromosome 21 and devoid of all endogenous genes is currently available.…”

“…Thus, HAC has been offered as an alternative vector in cell-mediated gene therapy, and therapeutic genes have been transferred into multiple stem cell types via HAC, for example, genomic human p53 into multipotent germline stem cell 7 and genomic Duchenne muscular dystrophy into mouse embryonic stem cell 5 and human and mouse induced pluripotent stem cells. 6 HACs support long-term correction of defective genes because expression from and transmission of these vectors are stable throughout many cell divisions in human cells. [4][5][6][7][8] This long-term stability is a significant improvement over the stability associated with current gene delivery methods.…”

“…6 HACs support long-term correction of defective genes because expression from and transmission of these vectors are stable throughout many cell divisions in human cells. [4][5][6][7][8] This long-term stability is a significant improvement over the stability associated with current gene delivery methods.…”