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Correction of Down syndrome and Edwards syndrome aneuploidies in human cell cultures

2015-08-31 00:01:13

OXFORD JOURNALS; 31 August 2015: DOI:10.1093/dnares/dsv016


Tomokazu Amano, Emiko Jeffries, Misa Amano, Akihiro C. Ko, Hong Yu, and Minoru S. H. Ko



Abstract


Aneuploidy, an abnormal number of chromosomes, has previously been considered irremediable. Here, we report findings that euploid cells increased among cultured aneuploid cells after exposure to the protein ZSCAN4, encoded by a mammalian-specific gene that is ordinarily expressed in preimplantation embryos and occasionally in stem cells. For footprint-free delivery of ZSCAN4 to cells, we developed ZSCAN4 synthetic mRNAs and Sendai virus vectors that encode human ZSCAN4. Applying the ZSCAN4 biologics to established cultures of mouse embryonic stem cells, most of which had become aneuploid and polyploid, dramatically increased the number of euploid cells within a few days. We then tested the biologics on non-immortalized primary human fibroblast cells derived from four individuals with Down syndrome - the most frequent autosomal trisomy of chromosome 21. Within weeks after ZSCAN4 application to the cells in culture, fluorescent in situ hybridization with a chromosome 21-specific probe detected the emergence of up to 24% of cells with only two rather than three copies. High-resolution G-banded chromosomes further showed up to 40% of cells with a normal karyotype. These findings were confirmed by whole-exome sequencing. Similar results were obtained for cells with the trisomy 18 of Edwards syndrome. Thus a direct, efficient correction of aneuploidy in human fibroblast cells seems possible in vitro using human ZSCAN4.



Introduction


Inborn deviations from the normal diploid cohort of 46 chromosomes, ‘aneuploidy’, causes severe problems in human development, growth, and function. Aneuploidy is caused by errors in chromosome segregation
during meiosis and mitosis, and is frequently found in cancer cells. The most common aneuploidy sustained in live births is trisomy 21 - three copies of chromosome 21 resulting in Down syndrome (DS). This complex developmental disorder involves congenital heart defects, haematopoietic disorders, early-onset Alzheimer’s disease, cognitive impairment, and premature ageing, including telomeres shorter than those of age-matched people without DS.



Although aneuploidy has conventionally been deemed irremediable, some researchers have recently aimed to develop ‘chromosome therapy’ for DS. One group has eliminated one copy of chromosome 21 by inserting a TK-NEO marker that can be selected against, and another has inactivated one copy of chromosome 21 by inserting
into it a copy of the XIST gene that normally inactivates one of the two X chromosomes in female cells. These procedures have thus provided proof of principle, but have not been applied to non-immortalized
primary cells due to their requirement of elaborate and cumbersome multistep genetic engineering. These limitations could be partially overcome by modifying induced pluripotent stem (iPS) cells from people with DS, but comparable culture and genetic manipulation before therapeutic intervention would still be required. A comparable limitation exists for a similar approach in which spontaneous correction of ring chromosomes occurs during the generation of human iPS cells from an individual with Miller - Dieker syndrome.



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