Fanconi anemia (FA) is an inherited disorder affecting DNA repair in hematopoietic (blood) stem and progenitor cells (HSPCs) from the bone marrow. As a consequence, blood cell counts are very low in patients and the immune system is severely weakened. Because of defects in DNA repair, FA patients are also at higher risk of developing cancer. Bone marrow transplantation coupled with gene therapy is a potential treatment, however, the paucity of available HSPCs and their sensitivity to ex vivo manipulation complicate the approach.
Drs. Jennifer Adair and Hans-Peter Kiem, in collaboration with Drs. Ann Woolfrey, Pamela Becker and Lauri Burroughs (Clinical Research Division) have been investigating the feasibility of gene therapy for FA patients in clinical trial. To improve isolation and modification of HSPCs as well as their engraftment in the transplant recipients, the authors modified recommendations from the international FA Gene Therapy Working Group. The results of their study were recently published in the journal Haematologica.
HSPCs are characterized by expression of the CD34 marker, which is used for cell enrichment followed by overnight transduction with a viral vector carrying the wild type FANCA gene, commonly mutated in FA. As explained by Dr. Adair, “our previous work and in research done by others in the field, there has been a clear need to correct the FA defect to elicit engraftment of FA cells. Whether these gene modified and corrected cells can provide a therapeutic benefit by regulating normal hematopoiesis in vivo or preventing the development of hematologic malignancy in FA patients remains to be seen, but preclinical data in mice and the rare incidence of somatic reversion, wherein the genetic defect in a FA patient acquires a corrected phenotype in as few as one blood cell clones, suggests that this is possible”.
However, the low frequency of CD34+ cells in FA patients limits the numbers of HSPCs that can be obtained by direct enrichment of CD34+ cells from the bone marrow. To limit loss of CD34+ cells, the procedure was modified to remove T and B lymphocytes, monocytes and granulocytes from bone marrow or mobilized peripheral blood leukapharesis cell products (ie. use of mobilization drugs to decrease HSPC retention in the bone marrow and allow their mobilization to the peripheral blood). This method of lineage depletion rather than cell enrichment limits CD34+ cell loss and manipulation.
When tested on healthy donor bone marrow cells, the lineage depleted cells and CD34+-enriched cells were transduced with similar efficiencies by the FANCA-carrying viral vector. According to Dr. Adair, “cells isolated by lineage depletion have lightly better output in the mouse xenograft model. This could be due to the lineage depletion process being more gentle on the CD34+ cells since it is an indirect enrichment rather than a direct enrichment. It could also be due to supporting cells in the graft, which are normally excluded when CD34+ cells are directly enriched.” The procedure was successfully replicated using mobilized peripheral blood leukapharesis from another FA patient. The purity of the CD34+ cells was higher than for the first two patients who underwent direct CD34 enrichment, and the transduction efficiency was similar.
When asked about future directions, Dr. Adair concluded: “Of course we are interested to see whether the enhanced engraftment observed in mice plays out in our patients. However, an ongoing limitation in FA gene therapy has been the inability to condition patients prior to gene therapy. One of the reasons for this is the lack of repopulating cells obtained in prior methods of isolation, such as CD34 enrichment, and the other major reason for this is the systemic toxicity of conditioning regimens which include DNA-damaging chemotherapy. We hope that this study will demonstrate the ability to not only isolate more of this limited stem cell pool from FA patients, but will also render these fewer cells more fit for engraftment. This could re-open the door for up-and-coming, non-chemotherapy based conditioning regimens currently under study to improve the therapeutic potential of gene therapy for the many FA patients who do not have suitable allogeneic donors available. We are also very excited to see whether this process can be extended to other diseases where CD34 content is limited, such as in sickle cell disease patients treated with hydroxyurea and patients with dyskeratosis congenital.”
Fred Hutch/UW Cancer Consortium faculty members Drs. Jennifer Adair, Hans-Peter Kiem, Pamela Becker, Lauri Burroughs and Ann Woolfrey.
Funding for this study was provided by a Sponsored Research Agreement between Fred Hutch and Rocket Pharmaceuticals, the Fred Hutch, the National Institutes of Health and National Cancer Institute.
Adair JE, Chandrasekaran D, Sghia-Hughes G, Haworth KG, Woolfrey AE, Burroughs LM, Choi GY, Becker PS, Kiem H-P. 2018. Novel lineage depletion preserves autologous blood stem cells for gene therapy of Fanconi anemia complementation group A. Haematologica. [Epub ahead of print].
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Maggie Burhans, Ph.D.
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Vaccine and Infectious Disease Division
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Julian Simon, Ph.D.
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