Can a faulty gene be saved?
Ever since the discovery in 1986 of the gene for dystrophin, the protein that’s missing in Duchenne muscular dystrophy (DMD), scientists and physicians have been trying to figure out how to compensate for its loss.
An obvious solution is to insert a new dystrophin gene, a technique usually referred to simply as gene therapy (see “Bridge Over Troubled Waters,” January-February).
But in the years since gene therapy experiments began, other ideas about how to compensate for errors in the dystrophin gene have arisen. These ideas range from repairing the gene, to modifying the way the cell interprets the language of the genetic code, to changing the activity of a gene that can code for a dystrophin substitute.
These techniques, based on the same genetic research that spawned the concept of gene therapy, can be loosely described as genetic modification. The following pages present four MDA-supported investigators who are among those who have begun to make genetic modification strategies a reality. While their focus is on Duchenne MD, if any of these techniques prove successful, it’s possible they could be applied to therapies for other neuromuscular diseases.
To understand the steps these investigators have taken, it helps to have a little knowledge of how the genetic code leads to the manufacture of proteins.
Breaking the code
Genes are made mostly of DNA, which is composed of nucleic acids attached to phosphate and sugar groups. When nucleic acids are attached to these other chemical groups, they’re called nucleotides.
|From gene to protein
One strategy to restore dystrophin production is gene repair, which aims to correct the dystrophin DNA sequence. Another is exon skipping, which involves changing the cutting and splicing mechanisms of the cell so that errors in the pre-mRNA are spliced out. Stop codon read-through likewise takes place at the RNA level, coaxing the cell to ignore a stop signal. Finally, utrophin upregulation ignores the dystrophin gene and attempts to boost production of the utrophin protein at any step from the utrophin DNA through protein synthesis.
The four nucleic acids in DNA are adenine (A), guanine (G), cytosine (C) and thymine (T), and when they’re arranged in specific sequences, they form a code that will ultimately determine the composition of protein molecules, which make up most cellular structures and carry out almost all cellular functions in the body.
Some nucleotide sequences are instructions for specific amino acids, the building blocks of proteins. Other sequences, known as stop codons, tell the cell’s mechanisms it’s time to stop reading the code.
Still others, splice sites, determine which parts of the genetic code will be reflected in the final protein’s components, and which parts will be cut out. The parts of the code that are destined to be cut out are known as introns, and the parts to be left in are called exons.
DNA is double-stranded, with the nucleic acids — A, G, C and T — stuck together between the strands like rungs of a ladder. The bonds between the strands are specific as well. Adenine is supposed to pair only with thymine, and guanine only with cytosine.
The first step in protein production is the building of RNA from DNA, a process called transcription.
RNA is very similar to DNA but differs in a few ways: It’s single-stranded; it contains the nucleic acid uracil (U) where DNA contains thymine (T); and its sugar groups aren’t exactly the same as DNA’s.
It’s from RNA that the final recipe for protein production will come, but not directly. RNA is first produced as a “rough draft,” known as pre-mRNA, and later edited to a shorter, final draft, known as messenger RNA or mRNA. The mRNA forms the template for final protein manufacture, known as translation.
At any stage in this process, errors can occur. But these stages also offer a possibility for either natural or laboratory-engineered correction.
Errors in the gene for dystrophin are usually one of two types. In deletions, parts of the coding sequence are missing. This leads to gaps in the RNA and then in the protein, and often interferes with the reading of otherwise correct information that follows the gap.
In the second type of error, point mutations, the wrong nucleic acid is inserted in place of the right one. Sometimes, point mutations cause premature stop codons, which tell the cell’s machinery to stop reading the genetic recipe before all the instructions have been read. A premature stop codon can be formed when only one nucleic acid is misplaced.
Fixing the code
One genetic modification strategy is DNA repair. It’s complete, it’s likely to be permanent, and it takes advantage of a natural cellular repair process. But so far, it’s been hard to get it to work well enough to be meaningful for people with Duchenne MD.
Another idea is coaxing the cell to “run a stop sign.” Chemical compounds that can cause “stop codon read-through” are under intense investigation, with clinical trials anticipated later this year.
Then there’s the possibility of changing the genetic code at the point at which the pre-mRNA has been made, with an error, but the final mRNA hasn’t yet been formed. Causing the cell to skip over the error-containing parts of the pre-mRNA and make a slightly shorter, but error-free, final RNA, can lead to a dystrophin molecule that’s highly functional. Known as exon skipping, this technique is gaining support.
Another tactic makes use of the fact that dystrophin has a near twin, a protein called utrophin that looks and acts very much like it but isn’t located in the same place in muscle cells and isn’t made in very large amounts. Increasing utrophin’s production and changing its location no longer seem far-fetched strategies to molecular biologists.