Scientists Combine Therapeutic Cloning, Embryonic Stem Cells, and Gene Therapy to Correct a Genetic Defect in Mice

CAMBRIDGE, Mass. — While the promise of nuclear transplantation therapy, commonly referred to as "therapeutic cloning," has given hope to patients, like Christopher Reeve, and excited the research community and the public, it has never been successfully demonstrated. Now, scientists from the Whitehead Institute for Biomedical Research have used a mouse model to establish for the first time that a combination of nuclear transplantation, gene therapy, and embryonic stem cell differentiation can be used to create custom-tailored cellular therapies for genetic disorders.

The work is a result of a collaboration between Whitehead Member Rudolf Jaenisch’s lab and Whitehead Fellow George Daley’s lab and will be published as two companion papers on the Cell web site on Friday, March 8, 2002.

The Whitehead researchers joined forces to work on a problem that until now has proven difficult to overcome. Scientists have been able to use nuclear transfer to create embryonic stem cells and differentiate them in culture to create many different cell types, including muscle, neurons, and hematopoietic stem, which are precursors to all immune and blood cells. But, they have never shown that the cells created in culture could be reintroduced into an animal to treat a disease.

Combining their independent research interests, the Jaenisch and Daley labs used skin cells from a mouse, which was completely immune deficient, to create a cellular therapy that was able to partially restore immune function in the mouse. "Though the immune system wasn’t completely restored, there was enough improvement to predict that a comparable result in humans would translate into a significant clinical benefit," says Daley.

"This is a proof-of-principle experiment, which shows that nuclear transplantation therapy may be possible for human application. Furthermore, it shows that gene therapy can be incorporated into the approach to correct genetic mutations in defective cells without affecting the germ line," added Jaenisch.

Essential Collaboration

Postdoctoral fellow William Rideout and graduate student Konrad Hochedlinger, both of the Jaenisch lab, used the nuclear transfer procedure to remove the nucleus, which contains the DNA of a cell, from an egg and replace it with the nucleus from a skin cell of an adult mouse with a genetic immune deficiency. In this procedure, the egg resets the developmental clock of the adult nucleus and the reprogrammed cell starts developing into an embryo that is genetically identical to the donor cell.

At the stage when the embryo develops into a hollow ball of approximately a hundred cells, called a blastocyst, it contains a nub composed of embryonic stem (ES) cells that have the potential to become any cell in the body. The ES cells from the blastocyst were isolated and the genetic defect causing the immune deficiency was corrected by gene therapy.

These corrected embryonic stem cells, however, couldn’t be successfully transplanted into the adult mouse to treat the immune disorder. For some reason, adult mice reject transplants of blood cell precursors derived from embryonic stem cells in culture.

"While embryonic stem cells could be induced to form hematopoietic cells in culture, these cells wouldn’t reliably generate the blood and immune system when transplanted into mice. For the last 15 years, engrafting mice with blood derived from embryonic stem cells has been the Holy Grail of the field," explained Daley.

Michael Kyba, a postdoctoral fellow in the Daley lab, found a way to achieve this goal by inserting a gene called HoxB4 that stimulates blood cell proliferation. The HoxB4 modified cells generated hematopoietic stem cell precursors that could be successfully transplanted into adult mice.

With this newfound ability, the researchers applied the same strategy to the genetically corrected embryonic stem cells made from the immunodeficient mouse. Remarkably, these genetically corrected cells were able to partially rescue the immune systems of mice suffering from complete immune deficiency.

Challenges

In principle, this approach might be useful some day for treating human patients with immune deficiency (ie. "bubble boy disease") or be applied to a host of other genetic diseases that can be corrected by cell transplantation. Embryonic stem cells can form any tissue in the body, including neurons, muscle cells of the heart, and pancreatic beta cells, which produce insulin.

In addition to the potential broad range of use, nuclear transplantation therapy to create embryonic stem cells has many benefits—the creation of cells that are genetically matched to the patient, the repair of genetic defects within cells to treat or cure inherited diseases, and the possibility of growing embryonic stem cells in culture for continued therapy as needed.

"Before the potential of nuclear transplantation therapy can be realized, much more research about the basic biology of stem cells has to done," says Daley. Unexpectedly, the researchers confronted interesting biological principles, which appear to be related to a fundamental difference between adults cells and cells derived from embryonic stem cells, even when the two types of cells are genetically identical. "While these results show nuclear transplantation therapy can work in principle, there are technical issues that we are working on now," explained Jaenisch.

Funding: The work from the Daley lab was supported by grants from the NIH, the National Science Foundation, MIT Biotechnology Process Engineering Center, the Leukemia and Lymphoma Society, the Canadian Institutes of Health Research, and the Alberta Heritage Foundation for Medical Research. The work from the Jaenisch lab was supported by Boehringer Ingelheim Fonds and the National Cancer Institute.

Citation

Rideout III, W. M., Hochedlinger, K., Kyba, M., Daley, G. Q., & Jaenisch, R. (2002). Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell, 109(1), 17-27.

Kyba, M., Perlingeiro, R. C., & Daley, G. Q. (2002). HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell, 109(1), 29-37.