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J.E. Adair et al.
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Figure 4. Multi-lineage engraftment of lineage depleted and transduced bone marrow (BM) and mobilized apheresis products (mAPH) in NSG mice. Recovery of total nucleated cells (TNC), CD34+ cells and lineage positive (+) cells (A and B). (C-E) Gene transfer efficiency. The colony-forming potential of transduced cells in standard CFC assays is defined as the plating efficiency (TNC). The colony-forming potential normalized to the number of CD34+ cells seeded is depicted as plating efficiency (CD34+). The percentage of colonies analyzed positive for the presence of lentivirus (LV) backbone by PCR analysis on DNA extracted from individual colonies is depicted as transduction efficiency. The vector copy number per cell in the bulk transduced population is depicted as VCN. The average VCN per cell in the individual CFC is depicted as single colony VCN. Data are representative of the average of 9 healthy BM products and 10 healthy mAPH products. Error bars rep- resent the standard error of the mean. (F) Engraftment of human CD45+ cells and lineage development into T cells (CD3+ ), monocytes (CD14+ ) and B cells (CD20+) was determined by flow cytometry over 20 weeks following infusion of lineage-depleted cell products. Data are representative of 36 mice from 6 mAPH donors and 42 mice from 6 BM donors, respectively. Error bars represent the Standard Error of the Mean.
tion on a blood product from an FA-A patient without compromising transduction efficiency.
Discussion
Here we confirm prior reports of inefficient CD34+ cell enrichment from FA patient blood products by direct, immunomagnetic bead-based separation, which is the current standard protocol for isolating HSPCs.15,19-21 We also demonstrate substantially reduced levels of CD34Hi cells in FA patients relative to healthy donors, which likely contributes to poor positive selection results in blood products from FA patients. Colony seeding assays demon- strate that only CD34Hi cells contribute to in vitro colony- forming potential in both FA and healthy donor blood products, underscoring the need to preserve as many available CD34+ cells as possible during ex vivo manipula- tion for gene transfer. We demonstrate a clinically viable procedure for depleting lineage positive cells to indirectly enrich for CD34+ cells that preserves the limited numbers of these cells in FA-A patients without compromising via- bility, gene transfer, or engraftment potential.
Importantly, the phenotype of limiting CD34+ cell num- bers is not restricted to FA alone. Sickle cell disease (SCD) patients treated with hydroxyurea also display reduced CD34+ cell frequencies in BM, and there is a contraindica- tion to mobilization of available CD34+ cells owing to an increased risk of vaso-occlusive crisis.22 Other inherited BM failure syndromes such as dyskeratosis congenita also
are associated with abnormal CD34+ cell frequencies and behavior.23 As a larger number of disease targets become relevant for gene therapy, additional patient populations will likely display variable CD34+ cell frequency and anti- gen expression. These disease targets could also benefit from clinically viable alternative selection procedures such as we have developed here.
Our observation of CFC potential in only the CD34Hi fraction in both FA and healthy samples suggests that CD34Lo cells may not be contributing to hematopoietic reconstitution. Notably, our data are from mAPH samples not BM, and we will need more patients for confirmation. Additionally, the standard colony-forming assay best defines progenitor cells, more so than true long-term repopulating hematopoietic stem cells.24 Alternatively, xenotransplant of purified cells into immunodeficient mice could provide the most robust evidence for CD34+ cell function in vivo, but the very small numbers of these cells may prove problematic to achieving relevant cell doses needed for these experiments. Another in vitro assay, such as the long-term culture-initiating cell assay,25 may provide additional insight into the desired target CD34+ subpopulations for gene therapy if they are present in either the CD34Hi or CD34Lo populations in FA patients. In this regard, we recently demonstrated that the CD34HiCD45RA–CD90+ phenotype is responsible for hematopoietic repopulation in non-human primates in the autologous, myeloablative setting,16 and evaluation of this phenotype in the enrolled FA patients is ongoing. Critically, our strategy of depleting cells expressing mature
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