Gene Targeting in Human ES Cells

Funding Type: 
SEED Grant
Grant Number: 
ICOC Funds Committed: 
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
The project is focused on developing efficient strategies to modify the genome of human embryonic stem cells (hESCs). Such modifications are likely to be essential for the full therapeutic potential of hESCs to be realized because they will allow for precise changes to be made in the hESC genome (without unwanted or unanticipated effects on the genome). Therapeutic changes made to the hESC genome could include those that correct inherited diseases, or those that augment the functions of critical genes that can confer a needed therapeutic effect. hESCs carrying changes of this sort can eventually be used in transplant settings to alleviate or cure diseases. The strategies favored by this project (those that depend on homologous recombination) have been widely exploited in model systems, but have yet to be optimized for use with hESCs. Various approaches will be tested in the project to optimize homologous recombination procedures for hESC genome modification. The cell lines generated in this project, the methodological expertise acquired, and the DNA reagents should all help to facilitate future efforts to generate hESCs carrying therapeutic mutations.
Statement of Benefit to California: 
The expertise we hope to gain in this project is essential for future projects that depend on generating precise changes in the human embryonic stem cell (hESC) genome. As has been the case with other experimental systems, once established, the technique of gene targeting by homologous recombination should be widely exploited for research and ultimately for therapeutic purposes. The State of California will benefit from this project because it will provide a means for realizing the full therapeutic and research potential that hESCs embody. Engineering specific changes into the hESC genome will allow for genes that cause disease to be corrected prior to the use of hESCs in transplant settings. It will also make a critical experimental tool available to researchers so that they can further understand biology of hESCs and thus make better use of the cells in therapeutic contexts.
Progress Report: 
  • Through this project, we have been successful in utilizing a gene therapy approach to deliver a newly discovered class of “small interfering RNA” (siRNA) to human stem cells. These siRNAs target and down-regulate proteins involved in recognition and rejection of donor cells by the immune system when they are transplanted into a recipient.
  • Initially it was thought that human embryonic stem cell (hESC)-derived cells and tissues might not be attacked by the immune system because these cells do not have Human Leukocyte Antigens on their surfaces in their primitive state. Human Leukocyte Antigens (HLA) are proteins that are expressed on the surface of almost all cells in the body. Because HLA sequences are highly variable and each person generally has a different set of HLA gene sequences, these cell surface markers serve as the identifiers of “self” vs. “non-self”. If immune cells in the body encounter foreign cells transplanted from a different individual, in most cases these foreign cells are recognized due to their display of a different “non-self” HLA on their cell surfaces, and attacked by the immune system. It is now known that once hESC start to develop into mature adult-type cells, they also start to increase their display of HLA, marking them as foreign “non-self” transplants. Thus, for hESC-derived cell and tissue transplants face the same problem of immune rejection as adult organ transplants.
  • To genetically reprogram human stem cells so they will not be rejected when transplanted into a different individual, we have used lentiviruses, a type of retrovirus which includes pathogens such as HIV, but as gene delivery vehicles (“vectors”), they have been completely disabled by removal of the viral genes, which have been replaced with the HLA-silencing siRNA sequences that we want them to deliver, thus turning viral foes into friends. Through this project, we have shown that this genetic reprogramming technique can be used to successfully down-regulate HLA expression in established human embryonic kidney cell lines, primary adult human blood-forming stem cells, primary adult human pluripotent stem cells, and human embryonic stem cells. We have also shown that, since the genetically re-programmed stem cells no longer display their own HLA due to siRNA-mediated silencing, they can no longer be recognized by immune cells or anti-HLA antibodies from other individuals, and are thus protected from immune rejection processes. We are proceeding to testing whether these genetically reprogrammed stem cells can evade the immune system when transplanted in vivo, and we have further developed a novel method for using chemotherapy to selectively expand the population of HLA-silenced stem cells after transplantation into the recipient.

© 2013 California Institute for Regenerative Medicine