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Obtaining blood stem cells for gene transfer in FA
A
B
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Figure 5. Multi-lineage engraftment levels of lineage-depleted cell products in NSG (NOD/SCID/IL2rgnull) mice is comparable to CD34-enriched cell products from the same donor. (A) Graph depicts percent recovery of total nucleated cells (TNC) and CD34+ cells from each arm following depletion or enrichment. (B) Numbers of total and transduced colony-forming cells (CFC) nor- malized to 1x108 cells processed to each arm, and vector copy number (VCN) in the bulk transduced cells following ten days of culture. Data are representative of 2 healthy donor bone marrow products. Error bars represent the Standard Error of the Mean. (C) Engraftment of human CD45+ cells and lineage development into T cells (CD3+), monocytes (CD14+) and B cells (CD20+) was determined by flow cytometry over 26 weeks following infusion. Data are representative of 9 mice for the lineage depleted (Lin-) arm and 6 mice for the CD34 enriched (CD34) arm, respectively.
Lin- CD34+
blood cell lineage markers preserves all CD34+ cell pheno- types for gene transfer and infusion, as demonstrated by Patient 3, whose infused CD34+ cell dose was the largest received to date.
One characteristic of lineage-depleted cell products requiring additional study is the presence and impact of other supporting cells on engraftment. Especially for BM- derived products, our procedure does not include a marker to deplete mesenchymal stem cells (MSC). While the engraftment potential of MSC manipulated ex vivo in CD34+ cell supportive media is unexplored, two recent reports suggest that these cells are integral to BM function in FA, and can be LV-transduced and functionally corrected to facilitate hematopoietic recovery and function in a mouse model of FA.26,27 For mAPH-derived products, such as that infused into Patient 3, additional follow up will be required to determine if a selective advantage is observed
in vivo. The improved transduction efficiency of lineage- depleted cell products could reflect non-repopulating CD34– cell uptake of LV. However, we still observed a ben- efit in transduction of hematopoietic CFC, even at the lower MOI of 5 IU/cell. One other possible explanation is the age and clinical condition of Patient 3. To address this concern we compared our results in Patient 3 to the 4 FA patients enrolled in the FANCOSTEM clinical trial in Spain (clinicaltrials.gov identifier: 02931071).28 These 4 patients were aged 3-7 years and demonstrated higher baseline blood cell counts at the time of collection. All 4 patients received the same mobilization regimen as Patient 3 reported here, but resulting mAPH products were subjected to direct CD34 enrichment prior to trans- duction at an MOI of 100 IU/cell. The reported mean VCN was 0.4±0.1 and ranged from 0.1 to 0.4 copies in individual CFC. Our data with a higher VCN at lower
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