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Scientists Cure Sickle Cell Anemia in Mice: Adult Skin Cells Coaxed to Produce Embryonic-like Properties

Published in UAB Insight, Winter 2008

A team of scientists from UAB and the Whitehead Institute for Biomedical Research have cured mice with humanized sickle cell anemia using a method that involves reprogramming skin fibroblasts to an embryonic-stem-cell-like state.

This process, which does not involve the use of embryos, is the first proof-of-principle in animals of a therapy using induced pluripotent stem (iPS) cells (Science. 2007;318:1920-1923).

The iPS cells have many of the same critical properties that have made embryonic stem cells the focus of intense research.

“The iPS cells express a number of the key genes expressed by embryonic stem cells, can be converted into virtually any cell type — brain, blood, lung, and liver, for example — and can divide to make more cells with the same attributes,” says UAB Department of Biochemistry and Molecular Genetics Chair Tim M. Townes, MD, coauthor of the Science paper.

“Like embryonic stem cells, iPS cells hold tremendous therapeutic promise. Once skin cells are converted to iPS cells, we can correct disease-causing mutations in vitro and transplant corrected cells back into the body where they start generating normal cells,” he says.

This scenario is precisely what the UAB/Whitehead Institute team achieved with the sickle cell mouse models, and Townes believes that adapting the technique for use in humans is only a matter of time.

The technique used to create iPS cells, which hold vast therapeutic potential, avoids the ethical storm surrounding embryonic stem cell work. “The process of generating iPS cells bypasses creation of a blastocyst that could potentially develop into a human being,” Townes says.

The iPS cells have another advantage over embryonic stem cells derived from fertilized eggs: Because tissue created with iPS cells is genetically identical to the recipient’s tissue, therapy based on this technique would avoid or much reduce the need for immunosuppression. “Without using cloning,” Townes says, “tissue created from embyronic stem cells would not be a genetic match for recipients.”

Curing Sickle Cell Anemia

As part of UAB’s 20-year search for a sickle cell anemia cure, scientists in Townes’ lab have developed transgenic knock-in mice with the full pathology of the human disease. “We replaced mouse globin genes with human globin genes. The mice make only human hemoglobin, and more importantly, human sickle hemoglobin. Curing the disease in these mice means a cure is possible in humans,” he says.

Townes and colleagues isolated skin cells from the tail tips of the mice and used retroviruses to introduce four genes expressing transcription factors that convert fibroblasts to iPS cells. The four transcription factors — Oct3/4, Sox2, c-Myc, and Klf4 — are known to be important regulators of stem-cell-like properties such as pluripotency.

Next, scientists “used an engineered construct created in the UAB lab and corrected the sickle mutation in iPS cells with gene-specific targeting,” Townes says. The cells were then transformed into hemapoietic stem cells and transplanted back into the diseased mice, where they began making red blood cells free from the sickle mutation.

“The animals’ sickle cell symptoms resolved completely, and at 4 months posttransplant they have not rejected transplanted cells, which are genetically identical to their own cells,” Townes says. He notes that although long-term monitoring is necessary, the cells appear self-renewing, and scientists expect them to produce all blood cell lineages for the animals’ lifetimes.

The paper by the UAB/Whitehead Institute team is the first to describe the successful use of iPS cells derived from a human disease model to correct a mutation and treat a disease. “These findings are a major step toward developing a cure for sickle cell anemia in humans,” Townes says. He and his team will continue focusing their iPS research on sickle cell anemia, working toward UAB’s long-held goal of curing the disease in humans.

Animals to Humans
Regenerative medicine strategies based on iPS cells are advancing rapidly.

Japanese scientist Shinya Yamanaka first described the revolutionary technique used by Townes and colleagues in 2006 (Cell. 2006;126:663-676). A little more than a year later, two labs, Yamanaka’s at Kyoto University and James A. Thomson’s at the University of Wisconsin — independently announced the successful reprogramming of human skin cells into iPS cells (Science. 2007;318:1920-1923) and (Cell. 2007;131:1-12).

To achieve the conversion, Yamanaka used the same four transcription factors that transformed skin fibroblasts into iPS cells in mice. Thomson, who more than a decade ago was the first to isolate human embryonic stem cells, substituted the genes NANOG and LIN28 for Klf4 and c-MYC. Like the tranformed mouse cells, iPS cells generated from human skin fibroblasts have virtually the same properties as embryonic stem cells.

Scientists must overcome several major issues before iPS-based therapy is ready for use in humans, however. A primary challenge is finding a safer delivery system for the transcription factors, Townes says.

“The retroviral vectors used to deliver the transcription factors into skin cells create too much unpredictability to use in humans. The retroviruses integrate randomly into the genome, where they could potentially disrupt genes and cause cancers,” he says. “A system for introducing these factors without insertion at random will have to be developed before we can move iPS therapy from animals to humans.” Townes’ group and others are investigating alternate delivery systems, including transiently expressed viruses and viruses that can be deleted.

Experts also are concerned about the reprogramming technique’s potential to stimulate the growth of tumors. One of the transcription factors used for conversion — c-Myc — is known to cause cancer. Mice treated with iPS cells reprogrammed with c-Myc are prone to tumors, as are their progeny.

Scientists are already tackling this problem. Yamanaka et al reported successful reprogramming of both human and mouse adult fibroblasts to iPS cells with a three-factor formula that does not include c-Myc (Nature Biotechnology. 2007;26:101-106).

The Future
Many public figures, including political and religious leaders who oppose embryonic stem cell research, are hailing iPS-based approaches to regenerative medicine as an end to the stem cell controversy and ultimately, as a potential solution to a number of devastating medical conditions.

David T. Curiel, MD, PhD, director of UAB’s Gene Therapy Center, says the stem cell story highlights one of the most fundamental debates in science — whether to marshal all available resources toward a single goal or to pursue more open-ended research that may produce answers to current questions a

s well as to those that have not yet been asked.

“With stem cells it is clear that we must be in the second category,” he says. “Stem cells represent a novel biological principle of enormous potential. Central to this is the recognition that intact adults possess pluripotential cells that can repopulate organs and offer the possibility for regeneration and repair. Yet, like every other revolutionary technology that has preceded it, the fruits are not likely to be realized quickly. It is possible that the diseases now thought to be the most approachable will not be easily solved and that applications that have not yet been considered will represent the first true success stories.”

The field of gene therapy has followed a parallel path. When it emerged on the scientific landscape more than 20 years ago, gene therapy promised “a wide array of medical possibilities,” Curiel says. “Although gene therapy has yet to deliver a clear genetic cure in a human interventional context, it has enabled many other novel technologies, including the transfer of genes that alters cells’ basic biology to one that is pluripotential and stem-like.”

Curiel and other experts question how soon iPS therapy can be made safe for direct therapy in humans. The regulatory hurdles involved in translating such work to humans are likely to be formidable, and iPS techniques may ultimately be most utilized for basic research — using the cells to discover human disease mechanisms and test novel drugs, for example.

Curiel is exploring various avenues of stem cell research. Last January he traveled to Spain to work with scientists at the Centro Nacional de Investigación Cardiovascular who are studying adult cardiac stem cells for myocardial regeneration. In addition, he and colleagues at UAB have proposed an investigation of mesenchymal stem cells as novel cell-delivery vehicles for therapy of ovarian cancer.

Many scientists strongly advise against abandoning human embryonic stem cell research in favor of an isolated focus on cell reprogramming techniques. Harvard University, for example, plans to continue its efforts to produce human embryonic stem cells through nuclear transfer.

“The iPS research that is now underway may be ahead of our ability to understand the impact of the technology, but that is the nature of science,” Curiel says. “This makes it critical to avoid focusing all stem cell efforts in a single direction.”

For more information:
Dr. Tim Townes
Dr. David Curiel