Gene editing has been able to silence the extra chromosome that is mostly responsible for Down syndrome (DS) in a cell-based study that could be the first step towards therapeutic treatment.
The approach uses a modified form of CRISPR-Cas9 to alter precise sections of DNA.
Researchers used this to insert the X-inactivation specific transcript (XIST) gene to deactivate X chromosomes in female cells, thereby correcting chromosomal triplication.
The partial transcriptional correction, reported in PNAS, offers a scalable, targeted platform for chromosomal therapy in Down syndrome and other aneuploidy disorders, which are conditions involving an abnormal number of chromosomes.
“These studies overcome a major hurdle in the treatment of Down syndrome (a genetic disorder cause by three rather than two copies of chromosome 21) by dramatically increasing the efficiency with which to insert XIST onto a single copy of chromosome 21 and thereby silencing this third copy,” senior researcher Volney Sheen, PhD, from Beth Israel Deaconess Medical Center, told Inside Precision Medicine.
“Determination of the best ways and times to deliver these constructs to the brain will become the next focus as we seek a clinical treatment for DS.”
Down syndrome is the most common genetic disorder and occurs in one out of 700 live births. It is linked with cognitive impairment, heart defects, and early-onset Alzheimer’s disease and results from the triplication of approximately 500 genes as well as other genetic changes on chromosome 21.
The XIST gene produces a long, noncoding RNA that inactivates many of the genes on one of the two X chromosomes of female mammals. It has therefore been mooted as a treatment for Down syndrome but technical limitations, including low levels of gene integration, have hindered progress.
In an attempt to address this, Sheen and team created a CRISPR-based method that involved fusing a codon-optimized λ-phage with Cas9, assembling single guide (sg)RNAs specific to single nucleotide polymorphisms (SNPs), and enhancing donor-acceptor DNA pairing.
The modified CRISPR-Cas9 method achieved a high level of genomic integration of large genetic material, improving the efficiency and specificity of XIST integration.
Inserting XIST onto one of the trisomic chromosome 21 alleles using SNP-dependent sgRNAs achieved an integration efficiency of 20% to 40% for the 14kb XIST gene.
XIST integration was revealed through expression of the enhanced green fluorescent protein reporter, clonal sequencing of individual lines, and fluorescent in situ hybridization.
The team further demonstrated that XIST activation led to upregulation of epigenetic markers, broad downregulation of messenger RNA expression on chromosome 21, and downregulation of specific genes on this chromosome.
“Our findings demonstrate atrial transcriptional correction of trisomic gene dosage and offer a scalable targeted platform for chromosomal therapy in DS and other aneuploidies,” the researchers reported.
They added: “The modified CRISPR method with XIST paves a rode for therapeutic treatment for DS.”
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