A new gene therapy strategy could transform the treatment of hundreds of rare genetic diseases. Photo credit: Sangharsh Lohakare via Unsplash.
New methods of gene therapy have the potential to treat hundreds of genetic diseases, all caused by the same type of error.
Genetic diseases…are caused by differences in DNA that lead to a huge range of symptoms, causing disability or death, and significantly impacting quality of life for those with the disease.
Genetic diseases are challenging conditions to treat. They are caused by differences in DNA that lead to a huge range of symptoms, causing disability or death, and significantly impacting quality of life for those with the disease. Many are caused by changes to DNA that convert important proteins into useless versions. Without the function of the important protein, the body is unable to carry out an essential process, resulting in symptoms depending on which process is affected.
One way in which proteins can be converted into useless versions is by shortening them. This can happen in response to specific mutations to DNA that cause protein fragments to be produced instead of complete versions.
The basis of 11% of genetic diseases
The central production line of biology uses DNA as a template to produce RNA, then uses this RNA to build proteins. During protein construction, the mRNA sequence is paired with recognition sites on tRNA molecules. Each tRNA carries an amino acid, and once these amino acids are stuck together, a protein is born. The mRNA is read in sequences of three, called codons. Each codon corresponds to a single tRNA, and therefore, a single amino acid.
Except three. There are three codons that tell the protein construction machinery when to stop extending a protein and to release it into the cell. These codons are essential for normal cell function, but random mutations can accidentally change normal codons into these “stop” codons. If this happens in the middle of a protein, then it gets released in an incomplete state—rendering it useless and resulting in genetic diseases such as class 1 cystic fibrosis.
The solution
There is a simple way to overcome these rogue stop codons: make a tRNA molecule that recognises them as normal codons instead. This lets cells complete the protein instead of releasing an incomplete fragment, despite the error in the codons. These tRNAs that can bypass stop codons are known as suppressor tRNAs, or sup-tRNAs.
Importantly, the efficiency of how well sup-tRNAs bypass erroneous stop codons is usually not important. The genetic diseases caused by poorly placed stop codons appear because cells have too few proteins to carry out a specific function. Even restoring a small number of these proteins is enough to effectively stop the disease.
It is reasonable to ask about the safety of sup-tRNAs, given that they bypass an important part of protein production. Nevertheless, cells have multiple safeguards that prevent this theoretical risk. Cells usually have multiple stop codons at points where they need protein construction to end, so a sup-tRNA that only recognises one is unlikely to cause any issues. Additionally, other molecules bind to the end of the codon chain, preventing sup-tRNAs from extending the protein any further. And if all else fails, the body destroys abnormal proteins that have been extended for too long. This explains the studies showing that cells continuing to live just fine after exposure to sup-tRNAs.
How to wield sup-tRNAs
Even if the science behind the treatment makes sense, basic logistical questions need to be answered before using it. As with most medical interventions, the main question is: how do we deliver it? Feeding sup-tRNAs to people or injecting them into the blood is unlikely to result in them being safely delivered to the right place.
A study from 2023 tried packaging sup-tRNAs inside lipid nanoparticles, which cells can then take up and release their contents. This showed encouraging results, as researchers could verify that cells used their sup-tRNAs in mice, and the tRNAs alleviated signs of cystic fibrosis in isolated human cells.
Meanwhile, another research group has used viruses to set up sup-tRNA factories inside cells. Recombinant adeno-associated viruses (rAAVs) are a modified form of virus with all the DNA required for replication removed. Instead, we add the DNA that we want cells to have; in this case, the DNA that encodes sup-tRNAs. When the virus infects cells, it gives them this DNA, letting them make sup-tRNAs on their own. This strategy was able to relieve the signs of a genetic disease (Hurler syndrome) in mice, and earlier this year the researchers expanded this system to target two out of the three stop codons.
Unfortunately, both systems have one main flaw. They require a lifetime of treatment. In the lipid nanoparticle packaging system, the body remains without the ability to make sup-tRNAs on its own, leaving it reliant on regular doses of nanoparticles. Meanwhile, rAAVs are not powerful enough. They only release DNA into cells rather than integrating it into the cell’s genome like other viruses, meaning that when new cells are born, they need to be given the DNA again. This could become even more difficult if the immune system starts recognising and destroying the viruses carrying this essential treatment.
A one-time treatment
To resolve this issue, Pierce et al. developed a third system, applying a recent innovation in gene editing called prime editing to integrate sup-tRNA production reliably into the genome. Instead of inserting a new piece of DNA into the genome, this method modifies an existing tRNA gene into a sup-tRNA gene. This is possible because cells have multiple copies of tRNA-producing genes, meaning that cell can survive losing one. They called this method ‘prime editing-mediated readthrough of premature termination codons’ (or PERT because that’s quite a mouthful).
…cells have multiple copies of tRNA-producing genes, meaning that cell can survive losing one.
After optimising the structure of the sup-tRNAs, refining the components used for gene editing, and carrying out the basic safety checks to ensure it didn’t do anything strange, they tested it out on a range of diseases caused by premature stop codons.
PERT sufficiently restored protein production levels in isolated human cells with Batten disease, Tay-Sachs disease, and Niemann-Pick disease type C1 to levels that should prevent symptoms. Additionally, treatment seemed to alleviate the symptoms of Hurler syndrome in another mouse model.
It doesn’t stop here
This progress doesn’t mean we’re close to curing all the genetic diseases that stop codons are responsible for. There are three different stop codons, and so far PERT has only been tested against one. Meanwhile, rAAVs have been used against two, but the third stop codon (UAA) has been challenging to identify a useable sup-tRNA for. Additionally, there’s the question of which amino acid to use in place of the stop codon; there’s 20 of them, and so far PERT has only been tested with leucine.
By treating multiple diseases at once, we can make it more likely that everyone gets the treatment they need
Nevertheless, these studies are a promising demonstration that it is possible to treat even the deepest diseases lurking in the human genome. This strategy is especially appealing because it could treat multiple rare diseases that, on their own, would be difficult to gain investment for when there are so many other pressing issues. By treating multiple diseases at once, we can make it more likely that everyone gets the treatment they need, not just those with the most common ailments.
Edited by Zohar Steinberg and Mathilda Lang.
