Editing genetic text

Only one man seems to have ever been cured of AIDS, a patient who also had leukemia. To treat leukemia, he received a bone marrow transplant in Berlin from a donor who, as luck would have it, was naturally immune to the AIDS virus.
If that natural mutation could be mimicked in human blood cells, patients could be endowed with immunity to the deadly virus. But there is no effective way of making precise alterations in human DNA.

That may be about to change, if a powerful new technique for editing the genetic text proves to be safe and effective. At the University of Pennsylvania, Dr Carl June and colleagues have used the technique to disrupt a gene in patients’ T cells, the type attacked by the AIDS virus. They have then infused those cells back into the body. A clinical trial is now under way to see if the treated cells will reconstitute a patient’s immune system and defeat the virus.

Zinc finger technique

The technique, which depends on natural agents called zinc fingers, may revive the lagging fortunes of gene therapy because it overcomes the inability to insert new genes at a chosen site. Other researchers plan to use the zinc finger technique to provide genetic treatments for diseases like bubble-boy disease, haemophilia and sickle-cell anaemia.

In principle, the zinc finger approach should work on almost any site on any chromosome of any plant or animal. If so, it would provide a general method for generating new crop plants, treating many human diseases, and even making inheritable changes in human sperm or eggs, should such interventions ever be regarded as ethically justifiable.

Zinc fingers are essential components of proteins used by living cells to turn genes on and off. Their name derives from the atom of zinc that holds two loops of protein together to form a “finger.” Because the fingers recognise specific sequences of DNA, they guide the control proteins to the exact site where their target gene begins.

After many years of development, biologists have learned how to modify nature’s DNA recognition system into a general system for manipulating genes. Each natural zinc finger recognises a set of three letters, or bases, on the DNA molecule. By stringing three or four fingers together, researchers can generate artificial proteins that match a particular site.

The new system has been developed by a small biotechnology company, Sangamo BioSciences of Richmond, Calif., and, to some degree separately, by academic researchers who belong to the Zinc Finger Consortium.

“We now have a full alphabet of zinc fingers,” Lanphier said, “but when we started the company it was like typing a novel with two fingers.” Zinc finger proteins have many potential uses. One is to link them to agents that turn on or turn off the gene at the site recognised by the fingers.

Ctrl C + Ctrl V of genetic text

More powerfully, the zinc fingers can be deployed as a word processing system for cutting and pasting genetic text. Two sets of zinc fingers are attached to a protein that cuts the DNA in between the two sites matched by the fingers. The cell quickly repairs the break but sometimes in a way that disrupts the gene. This is the approach used in destroying the gene for the receptor used by the AIDS virus to gain entry to white blood cells.

Or, if DNA for a new gene is inserted into a cell at the same time as the zinc fingers that scissor the DNA, the new gene will be incorporated by the cell’s repair system into the DNA at the break site. Most gene therapy techniques use a virus to carry new genes into a cell, but cannot direct the virus to insert genes at a specific site.

Zinc fingers can also be used for “trait stacking,” the positioning of several beneficial genes at a single site. This avoids heavy regulatory costs because genetically altered plants must be tested for safety for each site that is modified.

The zinc finger technology has taken many years to prepare because of the difficulty of designing the fingers and also of preventing them from cutting the genome in the wrong places. Only a handful of laboratories are currently using the technique, but proponents expect to see rapid growth.

The Zinc Finger Consortium, founded by Joung and Voytas, makes the method available free, and researchers need only pay for materials. But there are some 200 steps in Joung’s recipe for making zinc fingers, and it takes time and dedication to do them all correctly.

Resistant to HIV

The AIDS virus enters the T cells of the immune system by latching on to a receptor called CCR5, but about 10 per cent of Europeans have a mutation that disables the CCR5 gene. People who inherit two disabled copies of the gene do not have CCR5 on the surface of their T cells, so the AIDS virus has nothing to grab. These people are highly resistant to HIV.

In the zinc finger approach, the patient’s T cells are removed, and zinc finger scissors are used to disable the CCR5 gene. The treated cells are allowed to multiply, then reinjected into the patient. In experiments with mice, the treated cells turned out to have a strong natural advantage over the untreated ones, because those are under constant attack by the AIDS virus.

Whether or not zinc fingers will make gene therapy practical remains to be seen. “It’s a little too early to know since clinical trials are in their early stages,” said Dr Katherine A High, a haemophilia expert at the University of Pennsylvania.

Zinc fingers could be the gift that stem cell researchers have been waiting for. Stem cells taken from a patient may need to be genetically corrected before use, but until now there was no way of doing so.

Dr Rudolf Jaenisch, a stem cell expert at the Whitehead Institute in Cambridge, Mass., reported that he had successfully singled out three genes in induced embryonic stem cells with the help of zinc finger scissors designed by Sangamo.

Zinc fingers may also make technically possible a morally fraught procedure that has been merely a theoretical possibility, the alteration of the human germ line, meaning the egg or sperm cells. Genetic changes made in current gene therapy are to body cells, and they would die with the individual. But changes made to the germ line would be inherited. Many ethicists and others say this is a bridge that should not be crossed, since altering the germ line, even if justifiable for medical reasons, would lower the barrier to other kinds of change.