Dr. David W. Russell, assistant professor of medicine, and Roli Hirata, research technician at the University of Washington, report the successful use of a modified virus to perform a novel method of gene replacement that may be an important step toward overcoming obstacles to efficient gene therapy. Their findings are reported in the April issue of Nature Genetics.
Until now, gene therapy researchers have focused on gene addition, using a variety of modified viruses as vectors or transport vehicles to “infect” and insert the proper genetic material into cell nuclei that have genes with undesirable mutations. While the proper genetic material is inserted, it goes to random locations on the chromosome, and the faulty genetic material also remains.
By contrast, Russell and Hirata were able to achieve efficient gene correction. They succeeded in targeting the exact location of the mutated gene on the chromosome and replacing it with the correct genetic material present in the viral vector, at exactly the right location.
Russell uses a typewriter analogy: the new method finds the typographical error, whites it out and types in the proper sequence in the right place.
“If you had a ‘typo’ in a financial statement, say a missing zero, you’d want to fix it in the right place, not randomly anywhere in the statement,” he explains. “Or if you were correcting an instruction booklet with an error in it, you’d want to insert the corrected instruction in the right place on the right page.”
The UW researchers’ method guarantees that the new gene is controlled by appropriate genetic circuitry, ensuring that it is switched on in the right cells, at the right time and at the right dosage.
In addition to targeting exactly the right part of the cell, the repaired copy and the mutated copy of the genetic materal were exchanged at what researchers consider a very high frequency, in approximately 1 percent of targeted cells. The highest rates occurred in normal human fibroblasts — connective tissue cells.
“This level of exchange is perhaps 10,000 to 100,000 times better than has previously been achieved in normal human cells,” said Russell. “There are some diseases such as hemophilia that could perhaps be cured with only modest improvements in the gene correction rate. Many diseases could be cured if future research enables us to correct genes in 10 percent of the targeted cells.”
As a vector, the researchers used a virus called adeno-associated virus 2 (AAV), a single-stranded DNA virus capable of integrating into the chromosomes of mammals. Until this research, the potential of AAV vectors for gene targeting had not been explored. Russell and Hirata found that such vectors can target chromosomal genes at high frequencies and introduce modifications without creating additional mutations.
In an accompanying News & Views article, Dr. Alan Bernstein of the Samuel Lunenfeld Research Institute in Toronto states that the UW researchers’ achievement is an important step toward efficient gene therapy, and that their results, in combination with similar advances on other fronts, suggest that the technical obstacles to efficient, targeted replacement of defective genes will eventually be overcome.
The research was supported by grants from the American Society of Hematology, the Lucile P. Markey Charitable Trust, the March of Dimes Birth Defects Foundation and the National Institutes of Health.