FEATURE ARTICLE
Gene Therapy
Investigators have been searching for ways to add corrective genes to cells harboring defective genes. A better strategy might be to correct the defects
Eric Kmiec
Targeted Gene Repair
Ultimately, scientists would like to replace a dysfunctional gene with a functional one, within the normal context of the chromosome, an approach that could skirt the concerns about the number of genes delivered, the chromosomal location and the level of expression. Right now, homologous recombination, the only technique that comes close to this, is so inefficient that its success rate is 1 in 10,000. Needless to say, this is not adequate for human use.
But the idea of completely replacing a bad gene with a good one may be overreaching, especially when one considers how small are many of the mutations that contribute to disease. To understand how small, we must first consider a few basic facts about the composition of genes.
The gene is to inheritance what a word is to language; it is the basic unit of meaning. In the genetic lexicon, the gene is a length of DNA that codes for a particular protein. The alphabet used by the genetic language contains only five letters, or nucleotides, named for the bases. These are adenine (A), thymine (T), cytosine (C), guanine (G) and uracil (U). The nucleotides A, T, C and G are found in DNA. (RNA, a chemical cousin to DNA and an important participant in genetic decoding, lacks thymine, but has uracil in its place.) The average human gene is a little over 1,000 nucleotides long. In many inherited disorders only one or a few of these nucleotides is incorrect.
For example, sickle cell anemia is the result of a single nucleotide substitution, a single letter misspelled, in the gene encoding the b-globin strand of hemoglobin. Yet this one-nucleotide substitution can cause the structural deformity of the molecule and the characteristically distorted shape of the sickled red blood cell. Over 70 percent of the cases of cystic fibrosis are attributable to the deletion of three nucleotides in the CFTR gene. Why should the entire gene be replaced when the error is so minimal? That strategy seems akin to remodeling the whole kitchen to repair a leaky faucet.
In 1993, while studying homologous recombination in mammalian cells, members of my laboratory began experimenting with ways to repair damaged genes, rather than replacing them. The cell's own repair mechanisms are extremely efficient, as evidenced by the simple and continual inheritance of normal genes through generations of cell divisions. If we could harness the cell's own power of DNA repair, we reasoned, we might be able to correct mutations.
Normal human chromosomes are actually made up of two strands of DNA complexed to each other in an interesting way. It turns out that the nucleotides of DNA can bind with each other in a specific pattern. Except in very rare cases, adenine always pairs with thymine, and guanine always pairs with cytosine. Each DNA strand carries a nucleotide sequence exactly complementary to the other, such that every adenine nucleotide on one strand is matched up with a thymine on the partner strand, and every guanine is matched with a cytosine on the complementary strand. A sequence of GATC on one strand would therefore bind to the sequence of CTAG on its partner, or so it should be.
Occasionally the wrong nucleotide is inserted into a spot, so that the corresponding nucleotide on the partner strand cannot properly bind in that position. In that case the mismatched nucleotides form a bulge. Usually this is not a problem, since the cell contains DNA repair mechanisms that actually scan the DNA and detect such bulges. When one is discovered, the repair systems work to remove the incorrect nucleotide and replace it with the correct one. But if the mismatch is overlooked by the cell's repair machinery, the error is retained, and the gene remains defective.
It was our idea to alert these repair mechanisms to the error. The principle is quite simple. We artificially create a short string of nucleotides, called an oligomer, that, with one exception, is exactly complementary to the section of the gene in which the error is located, the exception being at the site of the error. Here we insert the nucleotide complementary to the one that is supposed to be in the DNA sequence of the normal gene. The oligomer binds to its complementary sequence on the DNA, and by design creates a bulge at the site of the mismatch. This bulge is eventually detected by the cell's internal DNA-repair mechanisms. Repair enzymes remove the erroneous nucleotide and replace it with a nucleotide complementary to the one in that position in the oligomer, which happens to be the correct nucleotide. This scenario for targeted gene repair has been experimentally confirmed in my laboratory by Allyson Cole-Strauss.
Our work builds on earlier studies from Fred Sherman's laboratory at the University of Rochester, who used a similar technique to change a single nucleotide. But the oligomers used by the Sherman group were unstable, and the team never extended its work. It turns out that mammalian cells contain enzymes that either degrade the ends of DNA molecules, or link them in long arrays called concatamers, which essentially destroys the integrity of the oligomers. We discovered that we could increase the stability of an oligomer by attaching segments of RNA to each of its ends. Like DNA, RNA is also composed of strings of nucleotides and therefore can bind to DNA in the same complementary manner as can another strand of DNA. (RNA contains no thymine. Instead, the uracil in RNA pairs with the adenine in DNA.)
In the past two years, we have successfully corrected seven chromosomal targets with this approach. Cole-Strauss and others in my lab have demonstrated the feasibility of using gene repair to correct the sickle-cell mutation in vitro, and Clifford Steer's laboratory has reproduced and extended those results in certain animal models. Vitali Alexeev and Kyggeon Yoon have shown that the correction is maintained through successive generations of cell division, suggesting that gene repair may have long-term benefits. Only continued studies will be able to determine whether this approach will be useful for human gene therapy.
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