Despite major advances in healthcare and disease prevention in the last hundred years, some of the most painful and serious non-infectious human diseases have eluded a cure. These are the so-called genetic diseases, in which a problem in a person’s genetic code causes the body to malfunction. Cystic fibrosis is a classic example, and it was the first genetic disease for which the exact genetic cause was identified. Other devastating genetic diseases include some muscular dystrophies, such as Duchenne Muscular Dystrophy, which typically kills patients before they reach their mid twenties.

The problem

The cells in your body contain the DNA that encodes the genetic information of the human genome. Part of the genome consists of functional units called genes. The sequence of DNA in a gene determines the gene’s function. Genetic diseases are caused by changes in the DNA sequence, called mutations.

A three-step process is required for a cell to convert the information contained in DNA into the protein molecules that do most of the cell’s work, such as metabolizing molecules to make energy or causing muscles to move. Proteins are made of long strings of molecules called amino acids, the sequence of which determines a protein’s structure and function. For proteins to do their jobs correctly, they must consist of the correct number and sequence of amino acid molecules.

The sequence of amino acids in a protein, in turn, is determined by the DNA sequence of its corresponding gene. DNA therefore contains the information required to produce proteins, but an additional molecule is needed to mediate the process of producing proteins from DNA. This mediating molecule is called RNA. One type of RNA, called messenger RNA (mRNA), is produced as a copy of the information encoded by DNA. The mRNA carries this information to regions of the cell where that information can be used to build proteins out of amino acids.

Because the mRNA is an exact transcription of its source DNA code, a mutated sequence of DNA will cause the resultant mRNA to contain the same mutation. When this mutated mRNA is subsequently used to produce a protein, the wrong number or sequence of amino acids will be incorporated into the finished protein. Depending on the nature of the mutation, this protein may not be able to do its job correctly, and may even cause disease. This is what happens in genetic diseases such as cystic fibrosis.

Current solutions

Intuitively, we could try to cure genetic diseases by directly correcting DNA mutations. This is called gene therapy, the act of directly altering a person’s DNA to fix mutated genes. However, there are many problems and ethical concerns with gene therapy. As DNA is contained in almost every cell in the body, mutated genes that cause genetic diseases are usually found in every cell too. Therefore, to fix a genetic disease one often needs to change the sequence of the mutated gene in every cell of the patient’s body. This could theoretically be accomplished by introducing artificially-made DNA to “replace” the mutated sequence. However, delivering the correct DNA to all the cells is extremely challenging, and is necessary for the therapy to be effective. Furthermore, it is difficult to get the DNA to integrate into the patient’s genome in exactly the right spot, to correct just the mutated gene without disturbing any of thousands of healthy genes. There are also concerns that the artificially introduced DNA could become a part of the DNA contained in the patient’s sperm or egg cells, and thereby be passed on to his or her children and grandchildren. Any problem with the inserted DNA, even if not immediately apparent, could possibly affect the descendants of the original patient in unforeseen ways.

A new approach

A paper published last month in the journal Nature suggests an alternative to classical gene therapy. John Karijolich and Yi-Tao Yu at the University of Rochester, New York showed that they could correct a particular type of genetic mutation without altering the DNA containing the mutation itself [1]. These experiments were done in yeast, which are among the most primitive organisms that share important cellular characteristics with humans – a group collectively referred to as eukaryotes. Because the work has yet to be performed on higher organisms, the medical potential of Karijolich and Yu’s findings is still a long ways away. However, the basic mechanics are of broad relevance and could open up incredible new possibilities for future medical advances.

Karijolich and Yu focused on a type of DNA mutation that causes too few amino acids to be incorporated into finished proteins. The DNA code consists of a linear sequence of bases. This code is copied into mRNA, and the protein-building machinery in cells “reads” the instructions encoded by the mRNA in clusters of three. These sets of three bases are called codons. Think of bases as letters, and codons as words.

Most codons instruct the machinery to add a specific amino acid to the growing chain that will eventually make up a mature protein. There are also special codons, called stop codons, that tell the machinery it has reached the end of the message and to cease adding amino acids to the protein. If the protein is complete, then this is the right thing to do. However, in around 11% of all genetic diseases [2], the DNA encoding the disease-causing protein is mutated to include a stop codon where none should be. When this sequence information is translated into a protein, a truncated protein is produced in which only the amino acid sequence up to the errant stop codon is made into protein, while the rest is omitted. Truncated proteins are usually unable to function properly, and production of these proteins can thus lead to disease.

The investigators at University of Rochester found that they could chemically alter a mutant mRNA containing an early stop codon. This alters the codon so that it is not read as a stop codon, but instead as a codon instructing the addition of an amino acid. The scientists accomplished this by converting uridine, the first base of the stop codon, into another molecule, pseudouridine. To achieve this, they used another type of RNA molecule, called H/ACA RNA. H/ACA RNA naturally targets mRNAs and carries out this chemical reaction in cells.

Figure 1. The DNA shown on the left contains a mutation (red block) that results in a truncated protein and disease. H/ACA RNAs can be used to fix the mutation at the mRNA step, allowing the normal “full-length” protein to be created. (Click image to enlarge.)

H/ACA RNA in action

The yeast gene that Karijolich and Yu chose to work on is called CUP1. When translated properly into the full-length protein, CUP1 allows yeast cells to grow in the presence of the chemical copper sulphate, which would normally kill them. When the researchers replaced normal yeast CUP1 DNA with a version containing an early stop codon, the yeast died in the presence of copper sulphate. This shows how the truncated CUP1 can have lethal effects on the yeast.

The researchers were then able to reverse this effect by targeting the CUP1 mRNAs containing the early stop codon with H/ACA RNA. The important thing to keep in mind is that the H/ACA RNA alters the messenger RNA, but not the DNA itself. So the mutated DNA code is unaltered, while the mRNA intermediary is changed. Thus, the H/ACA RNA converted the problematic “letter” in the mRNA from uridine to pseudouridine. As a result, the stop codon was no longer interpreted as a stop codon during protein synthesis. Instead, an amino acid was placed at that location. Furthermore, there is no longer a stop codon, so the rest of the mRNA is translated as well. The end result is the production of healthy, full length Cup1p.

Yeast whose mRNAs were altered by H/ACA RNA survived in the presence of copper sulphate just as well as yeast with the healthy CUP1 gene. Karijolich and Yu were thus able to treat the effects of a mutation in a yeast cell’s DNA without targeting the DNA directly. By causing the mRNA message produced from the DNA to be interpreted so as to produce a healthy, full-length protein, they circumvented the bad effects of the mutation and allowed the cells to survive.

Although yeast are quite distantly related to humans, they do share the basic DNA, mRNA, and protein production mechanisms with human cells. Yeast can therefore serve as a model for how these processes occur in humans. Because yeast live as single cells, it is easy to measure the production of proteins that have an effect on whether the yeast can live in certain environments. This makes yeast a good model that is much easier to work with at such early stages of research than humans or human cells.

Future work

Much additional research is still needed before this method can be transferred to use in human cells. Although human cells also have the H/ACA RNAs that were used by Karijolich and Yu in yeast, there may be differences in the way they function in humans. H/ACA RNAs target mRNAs through their sequences: their order of RNA “letters” or bases determines which mRNA they will bind to. The “rules” for how to target this binding have so far been worked out primarily for yeast [3], and are likely to be different in humans.

A further limitation of the current work is that Karijolich and Yu were not able to fully control which amino acid gets inserted in place of the stop codon in the “corrected” Cup1p protein. In some cases, one specific amino acid was inserted, but in others there was a 50/50 chance of getting one amino acid or another. For this to be useful in treating human disease, we will need to be able to introduce specific amino acids that do not disrupt the functionality of the protein product. Ideally, these will be the amino acids present in healthy versions of the fixed proteins. It is not clear from Karijolich and Yu’s work how difficult this may be. Finally, not all genetic diseases can be cured by this approach. Only the ~11% of diseases that are caused by early stop codon mutations can be directly addressed by this technique.

Another important issue to keep in mind is that human H/ACA RNAs may already be “correcting” mutations at the RNA level, so that mutations that might otherwise cause diseases are hidden. This however cannot logically be the case for mutations that do cause symptoms like those seen in cystic fibrosis or Duchenne Muscular Dystrophy. It seems likely that human H/ACA RNAs would need to be specially engineered to target mRNAs coding for mutated, disease-causing proteins, and this could be a serious technical challenge.

If engineered H/ACA RNAs can be designed, the next step will be to attempt a similar study to Karijolich and Yu’s work, using human cells grown in lab rather than yeast. If those experiments bear fruit, then the safety and efficacy of using targeted H/ACA RNAs in humans will need to be addressed. For therapies to work, engineered RNAs targeting disease-causing mRNAs will need to be introduced into patients in voluntary controlled trials. As in traditional gene therapy approaches, this will not be easy. The utmost care must be taken to ensure that the new therapy is effective in treating the disease while not being harmful to the patients.

Despite these problems, methods targeting human genetic diseases from another angle are likely to be welcomed, considering the difficulties encountered by classic gene therapy approaches. All disease therapies are the result of years – and often decades – of work, and targeted mRNA correction will be no exception. If this becomes an effective therapy, it is certain to prevent human suffering and will possibly save lives. That is a long way to go from making yeast cells live happily around copper sulphate, but it will be worth the journey.

Ruth McCole is a postdoctoral research fellow at Harvard Medical School.

Links of Interest

Muscular dystrophy:

Cystic fibrosis:

Gene therapy:


[1] Karijolich, J., & Yu, Y.-T. (2011). Converting nonsense codons into sense codons by targeted pseudouridylation Nature, 474(7351), 395–398. doi:10.1038/nature10165

[2] Mort, M., Ivanov, D., Cooper, D. N., & Chuzhanova, N. A. (2008). A meta-analysis of nonsense mutations causing human genetic disease Human mutation, 29(8), 1037–1047. doi:10.1002/humu.20763

[3] Ni, J., Tien, A. L., & Fournier, M. J. (1997). Small nucleolar RNAs direct site-specific synthesis of pseudouridine in ribosomal RNA Cell, 89(4), 565–573.

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