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Haematologica 2018 Volume 103(11):1806-1814
*JEA and H-PK contributed equally to this work.
Ferrata Storti Foundation
Bone Marrow Failure
Novel lineage depletion preserves autologous blood stem cells for gene therapy of Fanconi anemia complementation group A
Jennifer E. Adair,1,2* Devikha Chandrasekaran,1 Gabriella Sghia-Hughes,1 Kevin G. Haworth,1 Ann E. Woolfrey,1,2 Lauri M. Burroughs,1,2 Grace Y. Choi,1 Pamela S. Becker1,2 and Hans-Peter Kiem1,2*
Fred Hutchinson Cancer Research Center and 2University of Washington School of Medicine, Seattle, WA, USA
ABSTRACT
Ahallmark of Fanconi anemia is accelerated decline in hematopoi- etic stem and progenitor cells (CD34+) leading to bone marrow failure. Long-term treatment requires hematopoietic cell trans- plantation from an unaffected donor but is associated with potentially severe side-effects. Gene therapy to correct the genetic defect in the patient’s own CD34+ cells has been limited by low CD34+ cell numbers and viability. Here we demonstrate an altered ratio of CD34Hi to CD34Lo cells in Fanconi patients relative to healthy donors, with exclusive in vitro repopulating ability in only CD34Hi cells, underscoring a need for novel strategies to preserve limited CD34+ cells. To address this need, we developed a clinical protocol to deplete lineage+ (CD3+, CD14+, CD16+ and CD19+) cells from blood and marrow products. This process depletes >90% of lineage+ cells while retaining ≥60% of the initial CD34+ cell fraction, reduces total nucleated cells by 1-2 logs, and maintains transduction efficiency and cell viability following gene transfer. Importantly, transduced lineage– cell products engrafted equivalently to that of purified CD34+ cells from the same donor when xenotransplant- ed at matched CD34+ cell doses. This novel selection strategy has been approved by the regulatory agencies in a gene therapy study for Fanconi anemia patients (NCI Clinical Trial Reporting Program Registry ID NCI- 2011-00202; clinicaltrials.gov identifier: 01331018).
Introduction
Fanconi anemia (FA) is a rare monogenic disease with a wide array and variable presence of clinical symptoms, the hallmark of which is bone marrow (BM) failure.1 The genetic basis of FA is a mutation in any one of 21 genes2 whose protein com- ponents make up the FA/breast cancer pathway responsible for DNA repair of inter- strand crosslinks through nucleotide excision followed by homologous recombina- tion. Resulting compromises in genetic integrity are associated with a nearly uni- form decline in hematopoietic stem and progenitor cells (HSPCs), a 50% incidence of myelodysplastic syndrome or acute myeloid leukemia by adolescence, and a 25% lifetime incidence of head and neck squamous cell carcinoma or gynecological cancer.3 In some patients, blood cell clones demonstrate spontaneous reversion to wild type (i.e. somatic mosaicism) leading to improved and stable blood cell counts for up to 27 years.4-6 Thus, correction of the FA hematopoietic defect could signifi- cantly alter the disease’s clinical course, and this has driven decades of research in HSPC gene therapy for FA.
While FA was recognized as an early candidate disorder for gene therapy, several obstacles have been identified that have delayed clinical success.3 Initial clinical tri- als demonstrated a dramatic approximately 50-fold reduction in the number of true HSPCs in FA patients relative to other gene therapy patients, such as those treated for primary immune deficiencies.7 Moreover, FA HSPCs were exceptionally fragile when manipulated ex vivo for gene transfer. No treated patient has demonstrated
Correspondence:
hkiem@fredhutch.org or jadair@fredhutch.org
Received: April 6, 2018. Accepted: July 4, 2018. Pre-published: July 5, 2018.
doi:10.3324/haematol.2018.194571
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/103/11/1806
©2018 Ferrata Storti Foundation
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