For the first time, researchers have combined gene therapy and cellular reprogramming technologies in human cells to correct a genetic defect. After taking skin and hair cells from patients with a rare genetic disorder and fixing the aberrant mutation, the investigators successfully reprogrammed the cells to an embryonic-like state and then turned them into the very cell types that usually go awry, according to a study published online today (May 31) in Nature.

Stained FA-specific iPS cells Image: Juan Carlos Izpisúa Belmonte

The approach should be applicable to any disorder with simple, Mendelian inheritance, noted Juan Carlos Izpisúa Belmonte, a developmental biologist at the Salk Institute in La Jolla, Calif., and the Center for Regenerative Medicine in Barcelona, who led the study. "In principle," he wrote in an email, "our approach could be used with any disease that could be corrected by gene therapy and where there is loss or alteration of a specific cell type," not only for Fanconi anemia (FA), the disease his team focused on.

Over the past year, induced pluripotent stem (iPS) cells have been generated from patients with a wide variety of genetic disorders, including amyotrophic lateral sclerosis, spinal muscular atrophy, Parkinson's disease, and a handful of other pathologies. Such disease-specific stem cells offer unprecedented experimental models to investigate disease mechanisms and to screen new drug compounds. But to treat diseases with tailor-made cell therapies, such stem cells first need to be corrected to be disease-free. In 2007, the Whitehead Institute's Rudolf Jaenisch and the University of Alabama at Birmingham's Tim Townes used cellular reprogramming and gene targeting to correct and treat a mouse model of human sickle cell anemia. Now, Izpisúa Belmonte and his coworkers have accomplished a similar feat in human cells.

FA is a rare, recessive, bone marrow disorder caused by mutations in any of 13 genes. The researchers obtained skin and hair samples from three FA patients with mutations in two of these genes, and used lentiviral vectors to insert transgenic copies of the "normal" genes. They then used retroviruses containing the four "Yamanaka factors" to obtain 19 fully reprogrammed and repaired iPS cell lines that showed all the signatures of pluripotency. These cells expressed normal levels of the wild-type FA gene, and could be coaxed to form disease-free blood and bone marrow precursor cells.

Izpisúa Belmonte's team never succeeded in producing iPS cells without first correcting the genetic defects, however. The authors speculated that the diseased cells might not be reprogrammable because they had accumulated chromosomal abnormalities or continued to express mutated proteins at residual levels, but did not explore the issue further. M. William Lensch, a stem cell researcher at Children's Hospital Boston and Harvard Medical School who was not involved in the study, found this explanation unsatisfactory. "Why were the cells impossible to reprogram without being corrected in advance? That's what I really want to know," he told The Scientist. Teasing apart why the reprogramming techniques didn't work should help illuminate the underlying disease mechanics of FA, Lensch added.

Genetically-corrected fibroblasts (above) are reprogrammed to give rise to disease-free blood cell precursors (below) Image: Juan Carlos Izpisúa Belmonte

Townes also noted that the authors didn't characterize where exactly the FA transgene had integrated into the genome. This is important because the insertions could raise the specter of cancer. "If they interrupt a tumor suppressor, then you correct Fanconi anemia but the patient could get leukemia later," he told The Scientist, which is what happened in French gene therapy trials to treat X-linked severe combined immunodeficiency, in 2003.

The mended stem cells were not used to treat patients in the study. "In future it may become possible to transfer the corrected stem cells back into the patient, but much work remains to be done before this can be transferred from the lab bench to the bedside," said Chris Mathew, a molecular geneticist at King's College London, in a statement. The current reprogramming and gene therapy techniques -- which both involve introducing foreign DNA with potentially cancer-causing viruses -- are not suitable for therapeutic application, but researchers are working on developing safer reprogramming methods.

For example, earlier this week, Kwang-Soo Kim and his colleagues at Harvard Medical School reprogrammed healthy human fibroblasts without using any genetic materials; and, in March, a team led by Jaenisch created iPS cells from five Parkinson's disease patients using excisable viruses. Similar approaches combined with less hazardous gene therapy techniques, such as homologous recombination, could help move disease-corrected iPS cells into the clinic, Izpisúa Belmonte said. Indeed, Townes said he has unpublished data showing that he can fix human sickle cell samples using homologous recombination and reprogram the cells using "hit and run" vectors that don't leave a genetic trace.


Related stories:

Patient-ready iPS cells?
[28th May 2009]

A disease cell line library
[7th August 2008]

Diseased cells made pluripotent
[31st July 2008]