Xist. A potential gene therapy against Down syndrome?

By Themis Halka 

When talking about diseases arising from chromosomal abnormalities, Down syndrome is one which springs to mind. Caused by a trisomy of chromosome 21, it is the leading genetic cause of intellectual disabilities worldwide, as well as a comorbidity for multiple health issues, including hematopoietic disorders or early-onset Alzheimer’s.1

Chromosomes are the support of genes, and the regulation of their transcription in our cells is regulated by various mechanisms ensuring an appropriate and fine-tuned expression. Due to the additional chromosome 21 in the Down syndrome genome, the balanced expression of the genes located in this chromosome is disrupted, and this irregulation is the cause of major developmental problems. Numerous studies have aimed to determine the genes that, being overexpressed, participated in the manifestation of the disease, but for now, no specific gene has been found to be determinant in its development.1 It is believed that rather than the unbalanced dosage of particular proteins transcribed from a few genes, the proliferative disadvantage observed in the cell results from the collective slight over-expression of many genes.2

Therefore, if isolating genes that are harmful when overexpressed isn’t an appropriate target, would silencing the expression of the whole extra chromosome solve the issue? Is that possible? Jiang et al. (2013) have proposed an answer that would involve the X-linked gene Xist.3 Located on the X chromosome, Xist codes for a long non-coding RNA (lncRNA) that is  transcribed multiple times from only one of the X-chromosome in the female cells. This lncRNA coats its producing X-chromosome, preventing the binding of the transcription machinery, and thus the expression of the gene.4,5 This enables a co-ordinated expression of the X genes between females having two X chromosomes, and males having only one. Similarly, it was suggested that transfecting Xist to the third chromosome 21, preventing gene expression at the chromosomal level, could normalise Down syndrome by re-establishing a balance in the expression of genes carried by this chromosome3. Until then, the “genetic correction of the over-dose of genes across a whole extra chromosome in trisomic cells [had] remained outside the realm of possibility”.3 

Jiang et al. (2013) transfected Xist into induced pluripotent stem cells derived from trisomy 21 patient cells, precisely in the gene-rich core of chromosome 21, making sure it would be expressed.3 To achieve that, they used a genome editing technique, a zinc-finger nuclease (ZFN). ZFN are artificial endonucleases composed of a zinc finger protein and the cleavage domain of the restriction enzyme FokI.6 Zinc finger proteins are engineered to bind to particular domains of the DNA6, and allow the restriction enzyme to specifically cut the DNA at the targeted sequence.6,7 Then, the sequence of interest, in this case the Xist gene, can be inserted during DNA repair.7 Jiang et al. (2013) performed this site-specific addition of Xist in chromosome 21. According to measurements of the cells’ total chromosome 21, transcriptional output was reduced to near normal disomic levels, suggesting a successful repression of the genes of the third chromosome 21.3 

If this chromosome silencing has shown to be efficient to normalise transcription of the chromosome 21 genes, its effect on the underlying causes of Down syndrome phenotype can’t be assumed. In fact, the defects in cell function and pathogenesis might not be restored by gene silencing in cells that still carry the physical presence of an extra chromosome. Does the phenotype observed in Down syndrome solely arise from over-expression of the gene, or do other factors come into play? Would Down syndrome phenotypes be expressed in Xist-silenced trisomic cells? 

To investigate pathogenesis and cell function, Chiang et al. (2018) used human fetal hematopoiesis, the best characterized cellular phenotype in Down syndrome.8 Affected patients presenting more or less severe hematopoietic abnormalities, including increasing risks of developing lymphoblastic and acute megakaryocytic leukemias.9 Their investigation focused on human hematopoietic induced pluripotent stem cells (iPSCs), both comparing Xist and non-Xist silenced trisomic iPSCs, and contrasting obtained results with published data comparing human trisomic and disomic cells, as to measure the significance of gene silencing in trisomic iPSCs. The study revealed that the transgenic insertion of Xist in one of the chromosome 21 seemed to normalize the hematopoietic process, in particular the overproduction of megakaryocytes and erythrocytes, as well as an excess of CD43+ progenitors.8 Chiang et al. (2018) suggested that in affected cells, chromosome 21 silencing led to a normalization of the hematopoietic development, thanks to the rebalancement of dosage-sensitive chromosome 21 genes.8 

If other phenotypes of Down syndrome should be investigated, and Xist’s downstream effects analysed further, these studies are a major step in the development of gene therapy to fight Down syndrome phenotype development in trisomy 21 patients. In fact, the first step of any gene therapy approach, the possibility of correcting the genetic abnormality in vitro, has been successfully performed.8 Furthermore, gene editing techniques are constantly evolving, allowing ever more specificity in DNA breaking and sequence insertion, innovations that will increase the transgene’s probability of success as well as reducing off-target effects, ensuring minimal side effects on patients.


  1. Gardiner KJ. Molecular basis of pharmacotherapies for cognition in Down syndrome. Trends Pharmacol Sci. 2010;31(2): 66-73. Available from: doi:10.1016/j.tips.2009.10.010
  2. Blank HM, Sheltzer JM, Meehl CM, Amon A. Mitotic entry in the presence of DNA damage is a widespread property of aneuploidy in yeast. Mol Biol Cell. 2015;26(8): 1440-51. Available from: doi:10.1091/mbc.E14-10-1442
  3. Jiang J, et al. Translating dosage compensation to trisomy 21. Nature. 2013;500: 296–300. Available from: doi:10.1038/nature12394.
  4. Brown CJ, et al. The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus. Cell. 1992;71: 527–542. Available from: doi:10.1016/0092-8674(92)90520-m 
  5. Clemson CM, McNeil JA, Willard HF, Lawrence JB. XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J. Cell. Biol. 1996;132: 259–275. Available from: doi:10.1083/jcb.132.3.259.
  6. Urnov F, Rebar E, Holmes M et al. Genome editing with engineered zinc finger nucleases. Nat Rev Genet. 2010;11: 636–646. Available from: doi:10.1038/nrg2842
  7. Moehle EA, et al. Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc Natl Acad Sci U S A. 2007;104: 3055–3060. Available from: doi:10.1073/pnas.0611478104
  8. Chiang JC, Jiang J, Newburger PE, Lawrence JB. Trisomy silencing by XIST normalizes Down syndrome cell pathogenesis demonstrated for hematopoietic defects in vitro. Nat Commun. 2018;9(1): 5180. Available from: doi:10.1038/s41467-018-07630-y
  9. Banno K, Omori S, Hirata K, Nawa N, Nakagawa N, Nishimura K et al. Systematic Cellular Disease Models Reveal Synergistic Interaction of Trisomy 21 and GATA1 Mutations in Hematopoietic Abnormalities. Cell Rep. 2016 May 10;15(6): 1228-41. Available from: doi:10.1016/j.celrep.2016.04.031. 

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