Proof of Principle for Combined Cell and Gene Therapy for Fanconi Anemia

Optimal therapy for patients with genetic hematologic diseases would be to take the patient’s own cells, correct the genetic abnormality, and return the cells to the patient for long-term functional engraftment. Such therapy would require a combination of cell and gene therapy. In this paper, investigators from the laboratories of Juan Carlos Izpisúa-Belmonte in Barcelona and Juan Antonio Bueren in Madrid published proof-of-principle studies showing that cells from patients with Fanconi anemia (FA) can be genetically repaired and reprogrammed into inducible pluripotent stem (iPS) cells, which can then be guided to differentiate down the hematopoietic lineage in vitro.

FA is characterized by genomic instability and hypersensitivity to DNA damage. Patients generally present in childhood with anemia and/or susceptibility to infection and subsequently progress to complete bone marrow failure. Also, the increased susceptibility to DNA damage throughout the body increases the risk for developing solid malignancies. The iPS studies presented here present an option for repairing the hematopoietic system of these patients but not their other cells types. Bone marrow transplantation has been used to treat FA but is hampered by the risks of performing allogeneic transplantation as well as the high toxicity of chemotherapy in patients with FA.

iPS cells can be derived from skin fibroblasts and other dividing somatic cell types in vitro by introducing specific genes or gene products that reprogram the mature somatic cell nuclei so that the gene-expression pattern is modified to mirror that of embryonic stem (ES) cells. The most widely used current protocols use viral vectors to transfer four specific genes: OCT4, SOX2, KLF4, and c-MYC. Similar to ES cells, iPS cells can grow indefinitely in the laboratory and have the capacity, under specific in vitro conditions, to differentiate down every cellular lineage, including the hematopoietic lineage. In this work, the investigators were unable to reprogram skin fibroblasts from patients with FA into iPS cells unless they first “repaired” the genetic mutation by introducing a normal copy of the mutant FA gene. Cells were successfully reprogrammed only after the normal FA complex was restored; this teaches us new information regarding the genes required for iPS formation. The new iPS cell lines no longer showed a hypersensitivity to DNA-damaging agents. More importantly, hematopoietic cells derived from these patient-specific iPS cells also showed normal DNA repair in response to DNA-damaging agents.

There are significant hurdles to overcome before such an approach could be used therapeutically for humans with hematologic diseases. First, iPS cells will need to be safe. The goal is to develop iPS cells without permanent incorporation of the reprogramming genes (several of which are oncogenes), and to be able to differentiate the cells into entirely normal hematopoietic stem and progenitor cells that can reconstitute the hematopoietic system in the long term without any risk of malignancy. Investigators have succeeded in reprogramming adult somatic cells into iPS cells using either adenoviral vectors, which do not incorporate into the genome and eventually are lost, or with purified proteins that have been engineered to cross the cell membrane, and are thus only transiently available to reprogram the nucleus. However, even without permanent incorporation of the exogenous transgenes, the resultant iPS cells are still immortalized pluripotent cells that can form teratomas in vivo and can mutate in culture over time. Second, even though iPS can be differentiated into all types of blood cells in vitro, we do not yet know how to differentiate ES or iPS cells into normal, long-term, reconstituting hematopoietic stem cells.