|DBAF's Monthly Journal Club
Steven R. Ellis, PhD
DBAF Research Director
The latest in an increasingly long list of genes responsible for DBA has recently been published by Dr. Hanna Gazda and her colleagues in the Journal of Clinical Investigation1. Lo and behold, this is the first gene for DBA that does not encode a ribosomal protein. Instead, this gene encodes GATA1, a transcription factor involved in erythroid development. I will have more to say about GATA1 in subsequent Journals Clubs, but for now what this finding illustrates is the underlying genetic complexity of DBA and that they may be alternative pathways leading to the same disease state. This new finding raises the question of whether there are different forms of DBA, and if so, whether finding a cure for one form will be of benefit for another.
One potential cure that may not be dependent on the gene affected, is the possibility of using gene-corrected patient-derived induced pluripotent stem cells (IPSCs) to treat DBA patients. In an accompanying article in this month's newsletter, the DBAF has announced funding for Dr. Fred Goldman which will allow him to create and begin to study IPSCs from DBA patients.
A logical question at this juncture is, "What are IPSCs?" IPSCs are cells derived from a human subject that are genetically re-programmed to behave in a manner similar to embryonic stem cells, which you've probably heard discussed in the news and many other venues. Embryonic stem cells are cells created early during human development shortly after an egg is fertilized. These cells can differentiate into any cell type within the body and are referred to as being "pluripotent" to reflect this fact. The idea is that since these cells can differentiate into any other cell type, it might be possible to use these cells to replace cells that die either from old age, virus destruction, or even genetic diseases. There is considerable controversy around the use of embryonic stem cells, as they are derived from human embryos and so are construed by many as destroying human life, and so, morally objectionable as a therapeutic tool2.
This brings us back to the idea of using IPSCs as a surrogate for embryonic stem cells. These cells, which can be derived from cells found in human skin, can be re-programmed in a test tube back to a state where they behave much like an embryonic stem cell, and so could potentially be developed as cellular therapeutics without the same type of ethical qualms one encounters with embryonic stem cells. Moreover, by using cells derived from a patient to treat the same patient, it should be possible to avoid complications that arise from immunological rejection when transplanting organs or tissues from one individual to another.
The overly simplistic strategy for employing this technique as a cure for DBA is to take a patient's cells (let's say skin fibroblasts), re-program them to IPSCs, correct the underlying genetic defect, induce these pluripotent cells to become hematopoietic stem cells, and then transplant the patient with their own gene-corrected hematopoietic stem cells. The beauty here is that it really doesn't matter which gene is affected, as it is just a matter of correcting one gene rather than another (not that this is a trivial process). Of course, you must know the gene affected for this strategy to work, so consider this a shameless plug for ongoing gene discovery efforts in the DBA field.
So, what's the catch in all this, as it most certainly seems too good to be true? Well, such skepticism is warranted because at present there are still considerable obstacles to overcome before IPSCs become a therapeutic reality. The encouraging news however, is that because of its promise there are many groups working in this area on many disease fronts. In this regard, there are some very interesting papers recently published on Fanconi anemia field that illustrate where we may be going in applying this technology to DBA 3.
Fanconi anemia is another inherited bone marrow failure that differs from DBA in being caused by a defect in DBA repair rather than ribosome synthesis. Nevertheless, many of the principles involved in developing IPSCs for therapeutic uses in Fanconi anemia also apply to DBA. One of the challenges for creating IPSCs from Fanconi patients is that the re-programming process tends to activate p53, which also appears to be activated as part of the disease process. This double whammy, so to speak, in inducing p53 which in turn promotes cell death or arrests cell division, appears to reduce the frequency in which investigators are able to derive IPCSs from patient samples. We might expect some of the same challenges for DBA as p53 contributes to the disease process here as well 4. One strategy used by investigators in the Fanconi anemia field to get around this problem is to correct the genetic defect first in patient cells and then convert them to IPSCs. While this strategy has its own set of challenges, the paper by Müller and colleagues 3 and the accompanying commentary 5 illustrate that these challenges can be overcome and therefore bode well for efforts in creating IPSCs from DBA patients.
While progress is being made in resolving technical issues relating to developing IPSCs from certain patient populations, there are larger, more frightening, issues of safety that will need to be resolved before therapeutic use of IPSCs will become a reality. A good review on the promises and challenges of using IPSCs therapeutically has been published by Müller, Daley, and Williams 6.
One of the major concerns with IPSCs safety involves the re-programming process itself. When initially developed, it took the forced expression of 4 different genes to reprogram somatic fibroblasts to IPSCs. These genes were introduced into fibroblasts on viral vectors which integrate randomly into the human genome. This integration can activate certain genes or inhibit others and has been linked to cancer induction in certain gene therapy trials 7. Moreover, one of the four genes initially used in reprogramming is c-Myc, a major human oncogene, and so pose a significant cancer risk. It is now possible to reprogram cells without this oncogene thereby significantly reducing cancer risk. There are also strategies being developed to put all of the genes needed for re-programming on a single viral vector to limit the number of integration sites and further reduce the cancer risk. Another cancer risk is that when undifferentiated cells like IPSCs are injected into animals they create what are known as tetratomas, undifferentiated cells mass which have tumor like properties. So strategies are being developed to eliminate teratoma formation.
Even with reprogramming strategies becoming safer, there are additional obstacles to making therapeutic IPSCs a reality. Particularly relevant for DBA, is the fact that so far no one has been able to figure out how to create transplantable hematopoietic stem cells from IPSCs. I for one have complete faith in the creativity of the innumerable minds working on this problem and do not foresee it remaining a road block for too long.
Despite the fact that using IPSCs therapeutically in humans may still be a ways off, having IPSCs for experimental purposes will be a big boon for DBA research. These cells could be used to study the molecular underpinnings of DBA or as tools for drug screens. So let us wish Dr. Goldman all the best in his studies to create IPSCs from DBA patients, and look forward to the future and the myriad of ways that IPSCs can be used to find a cure.
1. Sankaran VG, Ghazvinian R, Do R, et al. Exome sequencing identifies GATA1 mutations resulting in Diamond-Blackfan anemia. J Clin Invest. 2012. Prepublished on 2012/06/19 as DOI 10.1172/JCI63597.
2. Lo B, Parham L. Ethical issues in stem cell research. Endocr Rev. 2009;30(3):204-213. Prepublished on 2009/04/16 as DOI 10.1210/er.2008-0031.
3. Muller LU, Milsom MD, Harris CE, et al. Overcoming reprogramming resistance of Fanconi anemia cells. Blood. 2012;119(23):5449-5457. Prepublished on 2012/03/01 as DOI 10.1182/blood-2012-02-408674.
4. Dutt S, Narla A, Lin K, et al. Haploinsufficiency for ribosomal protein genes causes selective activation of p53 in human erythroid progenitor cells. Blood. 2011;117(9):2567-2576. Prepublished on 2010/11/12 as DOI 10.1182/blood-2010-07-295238.
5. Papapetrou EP. FA iPS: correction or reprogramming first? Blood. 2012;119(23):5341-5342. Prepublished on 2012/06/09 as DOI 10.1182/blood-2012-04-417246.
6. Muller LU, Daley GQ, Williams DA. Upping the ante: recent advances in direct reprogramming. Mol Ther. 2009;17(6):947-953. Prepublished on 2009/04/02 as DOI 10.1038/mt.2009.72.
7. Baum C, Modlich U, Gohring G, Schlegelberger B. Concise review: managing genotoxicity in the therapeutic modification of stem cells. Stem Cells. 2011;29(10):1479-1484. Prepublished on 2011/09/08 as DOI 10.1002/stem.716.