Antisense oligonucleotides (ASOs) — short nucleic acid molecules used to modulate protein production — can be used to correct abnormal dopa decarboxylase (DDC) processing in cells from patients with aromatic L-amino acid decarboxylase (AADC) deficiency, a new study reports.
The study, “Antisense oligonucleotides modulate dopa decarboxylase function in aromatic l‐amino acid decarboxylase deficiency,” was published in Human Mutation.
Although it will be a very long time before such treatments might be available for AADC patients, further understanding the effects mutant genes have — and how these can be countered — continues to be a key mission for scientists as they seek therapies for this disorder.
AADC deficiency usually is caused by mutations in the DDC gene. Of these mutations, the most common, is called the DDC c.714+4A>T mutation (a conversion of 4 A residues to T residues at position 714). This mutation accounts for about 35 percent of AADC deficiency cases.
The researchers previously had delved into the exact mechanism by which this mutation decreases the levels of DDC enzyme that are produced, and they discovered this mutation leads to irregular splicing of DDC transcripts.
Splicing is the process by which parts of genes that are not supposed to be “read” for the final protein sequence are removed before the “reading” takes place. Most genes are composed of protein-coding regions (exons) and regions that don’t code proteins but often have other functions, like helping the gene “turn on or off” (introns).
During normal splicing of the DDC gene, the exons are usually stitched back together in order, e.g. 5-6-7-8. However, the researchers found that, in cells from AADC deficiency patients that carried the DDC c.714+4A>T mutation, the exons were often out of order. Specifically, exon 6 was often skipped or repeated, which leads to the wrong sequence being translated into a protein, ultimately decreasing the levels of the DDC enzyme.
In the new report, the researchers wondered whether they could find a way to stop this aberrant splicing. To do this, they used ASOs, which are small sequences of nucleotides that can bind to target sequences and help rectify splicing. Basically, the ASOs “blocked” the irregular cutting site, allowing normal splicing to restore itself.
Importantly, ASO molecules can be used in cells without interfering with the DNA itself, so they are fairly noninvasive as far as genetic engineering technologies go.
The researchers screened a number of ASOs to block the cutting site that repeated exon 6, and many of them had some effect; the most effective increased the proportion of normal DDC to 45%. However, none were able to restore levels to those seen in healthy control cells.
In addition, some of the ASOs had unintended effects, causing different aberrant splicing effects. Also, the ASOs did not affect the levels of molecules that skipped exon 6.
The researchers then tested whether their ASOs could increase the levels of DDC protein produced by cell lines derived from patients with AADC deficiency. Cells treated with the experimental ASOs had higher DDC protein levels than those treated with controls. ASO-treated cells also produced more serotonin, which the DDC enzyme helps to make.
“Our results further show that morpholino ASO treatments not only restored the normally spliced transcript 5-6-7-8 but also promoted the expression of DDC protein and serotonin in c.714+4A>T cells,” the researchers wrote.