Effectively tipping the balance in favour of the Mycobacterium surviving in the cell.

Autophagy: when ‘self-eating’ is good for you

Broken pieces of internal structures, damaged organelles and harmful clumps of proteins are all examples of the molecular detritus that builds up continuously in our cells. Fortunately, we are equipped with an intracellular process called autophagy (literally ‘self-eating’) that gathers up the debris, wraps it in a double membrane and delivers it to an intracellular sac called the lysosome. Here, the material is ingested, digested and recycled, ready to be used again.
Recent work has suggested that autophagy may also be important in killing intracellular bacteria that are able to escape the normal processes that control infection within cells. Autophagy appears to be critical in controlling infections of Mycobacterium tuberculosis (MTB) and related species called non-tuberculous mycobacteria (NTM), which are able to block degradation by lysosomes and thereby replicate within cells.
Researchers working at the University’s Cambridge Institute for Medical Research (CIMR) and Papworth Hospital have noticed that patients with chronic lung diseases such as cystic fibrosis (CF) are becoming increasingly infected with a highly pathogenic, multi-drug-resistant (MDR) NTM called Mycobacterium abscessus. Their research, published recently in the Journal of Clinical Investigation, suggests that M. abscessus infection may be linked to long-term use of azithromycin, an antibiotic with anti-inflammatory properties. They propose that azithromycin blocks autophagy in a type of white blood cell called the macrophage, effectively tipping the balance in favour of the Mycobacterium surviving in the cell.
It’s an ominous outcome for patients, as lead researcher Dr Andres Floto explained: “Developing an infection with Mycobacterium abscessus is a big deal for patients with CF. It’s resistant to virtually all antibiotics, is very hard to treat, can accelerate lung damage and may rule out future lung transplantation. While the benefits of long-term azithromycin therapy in CF are clear, our data suggest that there may also be a downside to watch out for.”
He added: “Recent studies showing the benefit of azithromycin therapy in asthma and smoking-related chronic obstructive pulmonary disease (COPD) will no doubt increase the numbers of patients on long- term azithromycin therapy. Physicians will need to be aware of the potential for harm with this treatment, and carefully monitor patients for mycobacterial disease”.

‘Forced stoppage’ of the cleaning crew

Floto is a Wellcome Trust Senior Clinical Fellow in the Department of Medicine and Principal Investigator in the CIMR. He is also Director of Research at the Cambridge Centre for Lung Infection at Papworth Hospital. The Centre cares for over 260 adults with CF and almost 2,000 patients with recurrent or difficult lung infections.
“We’d been struck by the rising rates of M. abscessus infection in CF patients in centres around the world and wondered whether it could be connected to an increased use of long-term azithromycin,” explained Floto. “Azithromycin is a broad-spectrum antibiotic which paradoxically is used to treat some mycobacterial infections. In patients with CF and, more recently, other inflammatory lung diseases such as asthma and COPD, it’s being prescribed as a long-term anti-inflammatory therapy.”
The association between azithromycin use and M. abscessus infection was first suggested when Floto and colleagues carried out an epidemiological study of adult patients with CF at Papworth Hospital and found that those patients with NTM infection were much more likely to be taking long-term azithromycin. When they looked at cells in culture, they discovered that the antibiotic impaired autophagy, effectively causing a ‘forced stoppage’ of the waste-recycling unit and preventing cells from clearing infecting mycobacteria. Moreover, azithromycin had a profound effect on M. abscessus infection of a mouse model; whereas untreated mice were able to clear the infection rapidly, those given the drug developed persistent lung infection.

Exploiting autophagy

The interactions of mycobacteria with immune cells are extremely complex and poorly understood. In a paper published in 2006 in Science, Floto together with Professor Paul Lehner at the CIMR began to define how immune cells respond to specific proteins from mycobacteria which then control the immune response. Subsequent work has suggested that the ability of macrophages to kill mycobacteria can be enhanced through a number of pathways, including autophagy.
Working with Professor David Rubinsztein at the CIMR, who has been interested in enhancing autophagy to clear the build-up of damaging clumps of proteins in neurodegenerative diseases, Floto has begun to study whether autophagy might be exploited therapeutically to treat multi-drug- resistant tuberculosis (MDR-TB), for which there is an urgent need to find new treatments. In 2008, the World Health Organization estimated that of the 9.4 million new TB cases 440,000 were MDR-TB. “It’s a massive challenge”, explained Floto, “We believe that stimulating autophagy to kill TB will bypass the problem of multi-drug resistance and may lead to potential new treatments for MDR-TB.”
Meanwhile, Floto’s work on azithromycin has already begun to influence clinical practice in a number of major CF centres in the UK. Although at pains to stress that a larger, prospective multi-centre study is needed before universal guidance can be given for the management of patients on chronic azithromycin therapy, Floto explained how the Cambridge Centre for Lung Infection has adopted a policy that is already proving successful: “We give patients who are not doing as well as we’d expect a holiday off the medication for a month while we screen carefully for NTM. When we do this, we often find a mycobacterial infection brewing in the lungs and can treat it immediately, instead of allowing the infection to take hold covertly without being detected.”
Floto’s research exemplifies the importance of a seamless link between fundamental research and clinical translation, and will benefit still further from the planned move of Papworth Hospital to the Cambridge Biomedical Campus . “Papworth has a unique group of patients and an international reputation for the treatment of patients with difficult lung infections,” he explained. “Forging greater links between Papworth and the University will inevitably promote research into a number of clinically important areas and smooth the route from bench to bedside, and back again.”

 http://www.cam.ac.uk/research/features/autophagy-when-self-eating-is-good-for-you/?source=researchnews

In New Way to Edit DNA, Hope for Treating Disease

Only one man seems to have ever been cured of AIDS, a patient who also had leukemia. To treat the 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.
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, hemophilia and sickle-cell anemia.

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 recognize 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 recognizes 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 biotech company, Sangamo BioSciences of Richmond, Calif., and, to some degree separately, by academic researchers who belong to the Zinc Finger Consortium.

Sangamo was founded in 1995 by Edward O. Lanphier II, a former executive with a gene therapy company. Reading an article by Aaron Klug, the British crystallographer who discovered the zinc finger design, he saw the technique’s potential for genetic manipulation. He bought a company Dr. Klug had founded and worked with him and researchers like Carl O. Pabo to improve the technique and develop combinations of zinc fingers to match any sequence of DNA letters.

“We now have a full alphabet of zinc fingers,” Mr. 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 recognized by the fingers.

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.

“I think it’s a broadly applicable technology which has already allowed experiments that would not have been possible before,” said J. Keith Joung, a biologist who designs zinc finger proteins at the Massachusetts General Hospital.

Daniel F. Voytas, a plant geneticist at the University of Minnesota, said the zinc finger technique would allow breeders to change the oil composition of any plant, the types of carbohydrates produced or the way carbon dioxide is captured. “We can go in and make any change we want to any plant species,” Dr. Voytas said.

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

The alternative is to buy zinc fingers. Sangamo has a commanding patent position and has licensed Sigma-Aldrich, a large life science company in St. Louis, to make zinc finger proteins for researchers. Sigma-Aldrich’s charge for a zinc finger protein that cuts the genome at the site of your choice is $39,000, with a discount for academic researchers. Zinc fingers that cut well-known human genes cost $12,000. Sigma-Aldrich has used the technology to generate rats with genetic defects that mimic human disease. A schizophrenic rat can be had for $100.

David Smoller, president of Sigma-Aldrich’s biotechnology unit, licensed the technology from Sangamo in 2006 when he felt the company had proved it worked. “This technology is just amazing,” Dr. Smoller said. “It’s a game changer.”

Sangamo has licensed the use of zinc fingers to Dow Agrosciences for creating new crop plants, and has reserved medical uses for itself. It has four Phase 2 clinical trials in progress, including treatments for diabetic neuropathy and amyotrophic lateral sclerosis.

In an ambitious effort to cure AIDS, Sangamo and the University of Pennsylvania started a clinical trial in February.

The AIDS virus enters the T cells of the immune system by latching on to a receptor called CCR5, but about 10 percent 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 H.I.V.

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, since 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 hemophilia expert at the University of Pennsylvania.

Dr. Matthew H. Porteus, a pediatric geneticist at the University of Texas, said, “I think it has the potential to solve a lot of the problems that have plagued the gene therapy field.” But Dr. Porteus noted that even the most carefully designed zinc fingers seemed to do some snipping away from their target site, a potentially serious safety problem.

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 had been no way of doing so.

Dr. Rudolf Jaenisch, a stem cell expert at the Whitehead Institute in Cambridge, Mass., reported in August that he had successfully singled out three genes in induced embryonic stem cells with the help of zinc finger scissors designed by Sangamo. “This is a really important tool for human embryonic stem cells,” Dr. Jaenisch said. The technology has not yet reached perfection. Some of the zinc fingers Sangamo provided “worked beautifully,” he said, but some did not.

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.

Several scientists were reluctant to discuss the issue, or dismissed it by saying that even zinc fingers did not meet the error-free standards that would be required for germ-line engineering. But zinc finger scissors are so efficient that only 5 to 10 embryos need be treated to get one with the desired result. This could make it practical to alter the germ line.

Since the germ lines of rats and zebra fish have already been altered with zinc finger scissors, “in principle there is no reason why a similar strategy could not be used to modify the human germ line,” Dr. Porteus said. The kind of disease that might be better treated in the germ line, if ethically acceptable, is cystic fibrosis, which affects many different tissues.
The disease could be corrected in unfertilized eggs, using the zinc finger technique, Dr. Porteus said. But he added, “I don’t think our society is ready for someone to propose this.”

http://www.nytimes.com/2009/12/29/health/research/29zinc.html?nl=health&emc=healthupdateema1&pagewanted=all